Combining radioimmunotherapy and antibody-drug conjugates for improved cancer therapy

ABSTRACT

Described herein are compositions and methods of use of radionuclide-antibody conjugates (for RAIT) and drug-antibody conjugates (ADC). The combination of RAIT and ADC was more efficacious than either RAIT alone, ADC alone, or the sum of effects of RAIT and ADC. The unexpected synergy resulted in decreased tumor growth rate and increased survival, with a high incidence of tumor-free survival in Capan-1 human pancreatic cancer xenografts in nude mice.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/855,213, filed Apr. 2, 2013, which was a divisional of U.S. patentapplication Ser. No. 12/957,655 (now issued U.S. Pat. No. 8,435,529),filed Dec. 1, 2010, which was a continuation-in-part of U.S. patentapplication Ser. No. 12/537,803 (now issued U.S. Pat. No. 8,491,896),filed Aug. 7, 2009; which was a continuation-in-part of U.S. patentapplication Ser. No. 11/849,791, filed Sep. 4, 2007, which was adivisional of U.S. patent application Ser. No. 10/461,885 (now issuedU.S. Pat. No. 7,282,567), filed Jun. 16, 2003. Those applicationsclaimed the benefit under 35 U.S.C. 119(e) of U.S. Provisional PatentApplication Ser. Nos. 60/388,314, filed Jun. 14, 2002; 61/087,463, filedAug. 8, 2008; and 61/144,227, filed Jan. 13, 2009. Ser. No. 12/957,655is a continuation-in-part of U.S. patent application Ser. No. 12/389,503(now issued U.S. Pat. No. 8,084,583), filed Feb. 20, 2009, which was acontinuation of U.S. patent application Ser. No. 11/745,896 (now issuedU.S. Pat. No. 7,517,964), filed May 8, 2007, which was a divisional ofU.S. patent application Ser. No. 10/377,121 (now issued U.S. Pat. No.7,238,785), filed Mar. 3, 2003, which claimed the benefit of U.S.Provisional Patent Application Ser. Nos. 60/360,229, filed Mar. 1, 2002.Ser. No. 12/957,655 is a continuation-in-part of U.S. patent applicationSer. No. 12/629,404 (now issued U.S. Pat. No. 7,999,083), filed Dec. 2,2009; which was a continuation of U.S. patent application Ser. No.12/026,811 (now issued U.S. Pat. No. 7,591,994); which was acontinuation-in-part of U.S. patent application Ser. No. 11/388,032,filed Mar. 23, 2006; which was a continuation-in-part of U.S. patentapplication Ser. No. 10/734,589 (now issued U.S. Pat. No. 7,585,491).Those applications claimed the benefit of U.S. Provisional PatentApplication Ser. Nos. 60/688,603, filed Apr. 6, 2005; 60/728,292, filedOct. 19, 2005; 60/751,196, filed Dec. 16, 1005; 60/433,017, filed Dec.13, 2002; and 61/207,890, filed Feb. 13, 2009. This application claimsthe benefit under 35 U.S.C. 119(e) of U.S. Provisional PatentApplication Ser. Nos. 61/266,356, filed Dec. 3, 2009; 61/292,656, filedJan. 6, 2010; 61/322,997, filed Apr. 12, 2010; and 61/323,952, filedApr. 14, 2010. The text of each priority application cited above isincorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 29, 2010, isnamed IMM324US.txt and is 21,844 bytes in size.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to combined therapy of cancer cells usingradiolabeled antibodies (RAIT) and antibodies conjugated to drugmoieties (ADC). In preferred embodiments, the combination of RAIT andADC exhibits a synergistic effect and is more effective to induce cancercell death than either RAIT or ADC alone, or the sum of the effects ofRAIT and ADC administered individually. In more preferred embodiments,the combination RAIT and ADC is effective to treat pancreatic cancer. Inmost preferred embodiments, the labeled antibodies or antibody fragmentsmay comprise a PAM4 (anti-pancreatic cancer mucin) antibody and an RS7(anti-Trop-2) antibody. However, the skilled artisan will realize thatother combinations of antibodies or fragments thereof may be utilizedand other forms of cancer may be treated.

2. Related Art

Pancreatic cancer is a malignant growth of the pancreas that mainlyoccurs in the cells of the pancreatic ducts. This disease is the ninthmost common form of cancer, yet it is the fourth and fifth leading causeof cancer deaths in men and women, respectively. Cancer of the pancreasis almost always fatal, with a five-year survival rate that is less than3%.

The most common symptoms of pancreatic cancer include jaundice,abdominal pain, and weight loss, which, together with other presentingfactors, are nonspecific in nature. Thus, diagnosing pancreatic cancerat an early stage of tumor growth is often difficult and requiresextensive diagnostic work-up, often times including exploratory surgery.Endoscopic ultrasonography and computed tomography are the bestnoninvasive means available today for diagnosis of pancreatic cancer.However, reliable detection of small tumors, as well as differentiationof pancreatic cancer from focal pancreatitis, is difficult. The vastmajority of patients with pancreatic cancer are presently diagnosed at alate stage when the tumor has already extended outside of the capsule toinvade surrounding organs and/or has metastasized extensively. Gold etal., Crit. Rev. Oncology/Hematology, 39:147-54 (2001). Late detection ofthe disease is common, and early pancreatic cancer diagnosis is rare inthe clinical setting.

Current treatment procedures available for pancreatic cancer have notled to a cure, or to a substantially improved survival time. Surgicalresection has been the only modality that offers a chance at survival.However, due to a large tumor burden, only 10% to 25% of patients arecandidates for “curative resection.” For those patients undergoing asurgical treatment, the five-year survival rate is still poor, averagingonly about 10%.

Antibodies, in particular monoclonal antibodies (MAbs) and engineeredantibodies or antibody fragments, have been widely tested and shown tobe of value in detection and treatment of various human disorders,including cancers, autoimmune diseases, infectious diseases,inflammatory diseases, and cardiovascular diseases [Filpula and McGuire,Exp. Opin. Ther. Patents (1999) 9: 231-245]. The clinical utility of anantibody or an antibody-derived agent is primarily dependent on itsability to bind to a specific targeted antigen associated with aparticular disorder. Selectivity is valuable for delivering atherapeutic agent, such as drugs, toxins, cytokines, hormones, hormoneantagonists, enzymes, enzyme inhibitors, inhibitory oligonucleotides(e.g., RNAi, siRNA), immunomodulators, radionuclides, anti-angiogenicagents or pro-apoptotic agents, to a targeted tumor. Radiolabeledantibodies have been used with some success in numerous malignancies,including ovarian cancer, colon cancer, medullary thyroid cancer, andlymphomas.

While various antibodies have been approved for human therapeutic use,including alemtuzumab, bevacizumab, cetuximab, gemtuzumab, ibritumomab,panitumumab, rituximab, tositumomab and trastuzumab, a need exists inthe field for more efficacious antibody-based therapies for difficult totreat cancers, such as pancreatic cancer.

SUMMARY

In various embodiments, the present invention concerns combinationtherapy with radiolabeled antibodies and drug-conjugated antibodies. Thecombination therapy may be of use for treatment of cancers for whichstandard therapies are not effective, such as pancreatic cancer. Inpreferred embodiments the combination of radiolabeled anddrug-conjugated antibodies is more efficacious than either radiolabeledantibody alone, drug-conjugated antibody alone, or the sum of theeffects of radiolabeled and drug-conjugated antibody administeredindividually. In specific embodiments, the antibodies may bindrespectively to human pancreatic cancer mucin and to EGP-1 (Trop-2).However, many antibodies against tumor-associated antigens (TAAs) areknown and the skilled artisan will realize that various combinations ofanti-TAA antibodies may be of use.

Preferably the antibodies of use bind specifically to cancer cells, withlittle or no binding to normal or non-neoplastic cells. More preferably,the antibodies bind to the earliest stages of cancer, such as PanIN-1Aand 1B and PanIN-2 in the case of pancreatic cancer. Most preferably,the antibodies bind to 80 to 90% or more of human invasive pancreaticadenocarcinoma, intraductal papillary mucinous neoplasia, PanIN-1A,PanIN-1B and PanIN-2 lesions, but not to normal human pancreatic tissue.

In a specific embodiment, the radiolabeled antibody may be a humanizedPAM4 antibody (see, e.g., U.S. Pat. No. 7,282,567, incorporated hereinby reference in its entirety), comprising the light complementaritydetermining region (CDR) sequences CDR1 (SASSSVSSSYLY, SEQ ID NO:1);CDR2 (STSNLAS, SEQ ID NO:2); and CDR3 (HQWNRYPYT, SEQ ID NO:3); and theheavy chain CDR sequences CDR1 (SYVLH, SEQ ID NO:4); CDR2(YINPYNDGTQYNEKFKG, SEQ ID NO:5) and CDR3 (GFGGSYGFAY, SEQ ID NO:6). Asdiscussed below, a number of therapeutic radionuclides of use for cancertreatment are known and any such known radionuclide may be conjugated tothe antibody of interest. In a more preferred embodiment, theradiolabeled antibody is ⁹⁰Y-hPAM4 (clivatuzumab tetraxetan).

In another specific embodiment, the drug-conjugated antibody may be ahumanized RS7 antibody (see, e.g., U.S. Pat. No. 7,238,785, incorporatedherein by reference in its entirety), comprising the light chain CDRsequences CDR1 (KASQDVSIAVA, SEQ ID NO:7); CDR2 (SASYRYT, SEQ ID NO:8);and CDR3 (QQHYITPLT, SEQ ID NO:9) and the heavy chain CDR sequences CDR1(NYGMN, SEQ ID NO:10); CDR2 (WINTYTGEPTYTDDFKG, SEQ ID NO:11) and CDR3(GGFGSSYWYFDV, SEQ ID NO:12). As discussed below, a number ofchemotherapeutic drugs of use for cancer treatment are known and anysuch known drug may be conjugated to the antibody of interest. In a morepreferred embodiment, the drug-conjugated antibody is SN-38-hRS7.

In alternative embodiments, the antibodies may be murine, chimeric,humanized or human antibodies that bind to the same antigenicdeterminant (epitope) as a PAM4 antibody comprising the lightcomplementarity determining region (CDR) sequences CDR1 (SASSSVSSSYLY,SEQ ID NO:1); CDR2 (STSNLAS, SEQ ID NO:2); and CDR3 (HQWNRYPYT, SEQ IDNO:3); and the heavy chain CDR sequences CDR1 (SYVLH, SEQ ID NO:4); CDR2(YINPYNDGTQYNEKFKG, SEQ ID NO:5) and CDR3 (GFGGSYGFAY, SEQ ID NO:6); orthat bind to the same epitope as an RS7 antibody comprising the lightchain CDR sequences CDR1 (KASQDVSIAVA, SEQ ID NO:7); CDR2 (SASYRYT, SEQID NO:8); and CDR3 (QQHYITPLT, SEQ ID NO:9) and the heavy chain CDRsequences CDR1 (NYGMN, SEQ ID NO:10); CDR2 (WINTYTGEPTYTDDFKG, SEQ IDNO:11) and CDR3 (GGFGSSYWYFDV, SEQ ID NO:12). Antibodies that bind tothe same antigenic determinant may be identified by a variety oftechniques known in the art, such as by competitive binding studies.

In preferred embodiments, the radiolabeled antibody and drug-conjugatedantibody are administered as separate antibody moieties, eithersequentially or concurrently. However, in alternative embodiments, theradiolabeled antibody and drug-conjugated antibody, or fragmentsthereof, may be administered as a bispecific antibody. In preferredalternative embodiments, the bispecific antibody may be produced by thedock-and-lock (DNL) technique, as discussed in more detail below.

Although in preferred embodiments, the antibodies or antibody fragmentsare conjugated respectively to a radionuclide and a chemotherapeuticdrug, the skilled artisan will realize that other anticancer therapeuticagents are known in the art and may potentially be substituted for, orused in addition to, the subject radionuclide and/or drug. Other knowntherapeutic agents include toxins, immunomodulators (such as cytokines,lymphokines, chemokines, growth factors and tumor necrosis factors),hormones, hormone antagonists, enzymes, oligonucleotides (such as siRNAor RNAi), photoactive therapeutic agents, anti-angiogenic agents andpro-apoptotic agents. The therapeutic agents may comprise one or morecopies of the same therapeutic agent or else combinations of differenttherapeutic agents. The therapeutic agents may be conjugated to thesubject antibodies or separately administered before, concurrently withor after the subject antibodies.

In a preferred embodiment, the therapeutic agent is a cytotoxic agent,such as a drug or a toxin. Also preferred, the drug is selected from thegroup consisting of nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas, gemcitabine, triazenes, folic acid analogs,anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purineanalogs, antibiotics, enzyme inhibitors, epipodophyllotoxins, platinumcoordination complexes, vinca alkaloids, substituted ureas, methylhydrazine derivatives, adrenocortical suppressants, hormone antagonists,endostatin, taxols, camptothecins, SN-38, doxorubicins and theiranalogs, antimetabolites, alkylating agents, antimitotics,anti-angiogenic agents, tyrosine kinase inhibitors, mTOR inhibitors,heat shock protein (HSP90) inhibitors, proteosome inhibitors, HDACinhibitors, pro-apoptotic agents, methotrexate, CPT-11, and acombination thereof.

In another preferred embodiment, the therapeutic agent is a toxinselected from the group consisting of ricin, abrin, alpha toxin,saporin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A,pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonasexotoxin, and Pseudomonas endotoxin and combinations thereof. Or animmunomodulator selected from the group consisting of a cytokine, a stemcell growth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), erythropoietin,thrombopoietin and a combinations thereof.

In other preferred embodiments, the therapeutic agent is a radionuclideselected from the group consisting of ¹¹¹In, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, ²¹¹At,⁶²Cu, ⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm,¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe,⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au,¹⁹⁹Au, and ²¹¹Pb. Also preferred are radionuclides that substantiallydecay with Auger-emitting particles. For example, Co-58, Ga-67, Br-80m,Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, 1-125, Ho-161, Os-189m andIr-192. Decay energies of useful beta-particle-emitting nuclides arepreferably <1,000 keV, more preferably <100 keV, and most preferably <70keV. Also preferred are radionuclides that substantially decay withgeneration of alpha-particles. Such radionuclides include, but are notlimited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211,Ac-225, Fr-221, At-217, Bi-213 and Fm-255. Decay energies of usefulalpha-particle-emitting radionuclides are preferably 2,000-10,000 keV,more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.Additional potential radioisotopes of use include ¹¹C, ¹³N, ¹⁵O, ⁷⁵Br,¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ¹¹³In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru, ¹⁰⁷Hg,²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁹⁷Pt,¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵⁸Co, ⁵¹Cr, ⁵⁹Fe,⁷⁵Se, ²⁰¹Tl, ²²⁵Au, ⁷⁶Br, ¹⁶⁹Yb, and the like. In other embodiments thetherapeutic agent is a photoactive therapeutic agent selected from thegroup consisting of chromogens and dyes.

Alternatively, the therapeutic agent is an enzyme selected from thegroup consisting of malate dehydrogenase, staphylococcal nuclease,delta-V-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. Such enzymes may be used, for example, incombination with prodrugs that are administered in relatively non-toxicform and converted at the target site by the enzyme into a cytotoxicagent. In other alternatives, a drug may be converted into less toxicform by endogenous enzymes in the subject but may be reconverted into acytotoxic form by the therapeutic enzyme.

In other alternative embodiments, anti-TAA antibodies other than PAM4and/or RS7 may be utilized. Preferably, the antibody or fragment thereofbinds to a tumor-associated antigen selected from the group consistingof CA19.9, DUPAN2, SPAN1, Nd2, B72.3, CC49, CEA (CEACAM5), CEACAM6,Le^(a), the Lewis antigen Le(y), CSAp, insulin-like growth factor(ILGF), epithelial glycoprotein-1 (EGP-1), epithelial glycoprotein-2(EGP-2), CD80, placental growth factor (PlGF), carbonic anhydrase IX,tenascin, IL-6, HLA-DR, CD40, CD74 (e.g., milatuzumab), CD138(syndecan-1), MUC-1, MUC-2, MUC-3, MUC-4, MUC-5ac, MUC-16, MUC-17,TAG-72, EGFR, platelet-derived growth factor (PDGF), angiogenesisfactors (e.g., VEGF and PlGF), products of oncogenes (e.g., bcl-2, Kras,p53), cMET, HER2/neu, and antigens associated with gastric cancer andcolorectal cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. PET/CT fusion images for a patient with inoperable metastaticpancreatic cancer treated with fractionated ⁹⁰Y-hPAM4 plus gemcitabine,before therapy (left side) and post-therapy (right side). The circleindicates the location of the primary lesion, which shows a significantdecrease in PET/CT intensity following therapy.

FIG. 2. 3D PET images for a patient with inoperable metastaticpancreatic cancer treated with fractionated ⁹⁰Y-hPAM4 plus gemcitabine,before therapy (left side) and post-therapy (right side). Arrows pointto the locations of the primary lesion (on right) and metastases (onleft), each of which shows a significant decrease in PET image intensityafter therapy with radiolabeled hPAM4 plus gemcitabine.

FIG. 3. Therapeutic activity of a single treatment of established (˜0.4cm³) CaPan1 tumors with 0.15 mCi of ⁹⁰Y-hPAM4 IgG, or 0.25 or 0.50 mCiof TF10-pretargeted ⁹⁰Y-IMP-288.

FIG. 4. Effect of gemcitabine potentiation of PT-RAIT therapy.

FIG. 5. Effect of combination of cetuximab with gemcitabine and PT-RAIT.

FIG. 6. Therapeutic efficacy of (Q)-hRS7 demonstrated in a Calu-3 humanxenograft model to inhibit tumor growth (A) and increase MST (B). Nudemice were inoculated subcutaneously with 1×10⁷ Calu-3 cells. When tumorsreached approximately 0.15 cm³, mice were treated with either a singleintravenous dose of 50 μg or two injections of 25 μg administered sevendays apart. Control animals received saline.

FIG. 7 illustrates (A) the structure of bifunctional SN-38 (CL2A-SN-38);(B) a synthetic scheme for preparing CL2A-SN-38; and (C) a conjugationscheme for attaching CL2A-SN-38 to an antibody.

FIG. 8 compares the therapeutic efficacy of hRS7-SN-38 alone, ⁹⁰Y-hPAM4alone, and the combination of hRS7-SN-38 and ⁹⁰Y-hPAM4, at either 75 μCior 130 μCi.

FIG. 9 illustrates the toxicity, measured as % weight loss, of RAITalone and ADC alone versus the combination of RAIT plus ADC.

FIG. 10 compares the effects of simultaneous RAIT and ADC withsequential administration of the two treatments.

FIG. 11 shows the effects of RAIT and ADC performed with the same(hPAM4) antibody.

FIG. 12 indicates the comparative efficacies of different antibodyconjugates of SN-38.

DETAILED DESCRIPTION Definitions

Unless otherwise specified, “a” or “an” means one or more.

As used herein, “about” means plus or minus 10%. For example, “about100” would include any number between 90 and 110.

An antibody, as described herein, refers to a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment.

An antibody fragment is a portion of an antibody such as F(ab′)₂, Fab′,Fab, Fv, sFv and the like. Regardless of structure, an antibody fragmentbinds with the same antigen that is recognized by the full-lengthantibody. The term “antibody fragment” also includes isolated fragmentsconsisting of the variable regions of antibodies, such as the “Fv”fragments consisting of the variable regions of the heavy and lightchains and recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“scFvproteins”).

A chimeric antibody is a recombinant protein that contains the variabledomains including the complementarity determining regions (CDRs) of anantibody derived from one species, preferably a rodent antibody, whilethe constant domains of the antibody molecule are derived from those ofa human antibody. For veterinary applications, the constant domains ofthe chimeric antibody may be derived from that of other species, such asa cat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a rodent antibody, are transferred fromthe heavy and light variable chains of the rodent antibody into humanheavy and light variable domains (e.g., framework region sequences). Theconstant domains of the antibody molecule are derived from those of ahuman antibody. In certain embodiments, a limited number of frameworkregion amino acid residues from the parent (rodent) antibody may besubstituted into the human antibody framework region sequences.

A human antibody is, e.g., an antibody obtained from transgenic micethat have been “engineered” to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain loci are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous murine heavy chain and light chain loci. Thetransgenic mice can synthesize human antibodies specific for particularantigens, and the mice can be used to produce human antibody-secretinghybridomas. Methods for obtaining human antibodies from transgenic miceare described by Green et al., Nature Genet. 7:13 (1994), Lonberg etal., Nature 368:856 (1994), and Taylor et al., Int. Immun. 6:579 (1994).A fully human antibody also can be constructed by genetic or chromosomaltransfection methods, as well as phage display technology, all of whichare known in the art. See for example, McCafferty et al., Nature348:552-553 (1990) for the production of human antibodies and fragmentsthereof in vitro, from immunoglobulin variable domain gene repertoiresfrom unimmunized donors. In this technique, antibody variable domaingenes are cloned in-frame into either a major or minor coat protein geneof a filamentous bacteriophage, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. In this way, the phage mimics some of the properties of theB cell. Phage display can be performed in a variety of formats, forreview, see e.g. Johnson and Chiswell, Current Opinion in StructuralBiology 3:5564-571 (1993). Human antibodies may also be generated by invitro activated B cells. See U.S. Pat. Nos. 5,567,610 and 5,229,275, theExamples section of which are incorporated herein by reference.

A therapeutic agent is a compound, molecule or atom which isadministered separately, concurrently or sequentially with an antibodymoiety or conjugated to an antibody moiety, i.e., antibody or antibodyfragment, or a subfragment, and is useful in the treatment of a disease.Examples of therapeutic agents include antibodies, antibody fragments,drugs, toxins, nucleases, hormones, immunomodulators, pro-apoptoticagents, anti-angiogenic agents, boron compounds, photoactive agents ordyes and radioisotopes. Therapeutic agents of use are described in moredetail below.

An immunoconjugate is an antibody, antibody fragment or fusion proteinconjugated to at least one therapeutic and/or diagnostic agent.

A multispecific antibody is an antibody that can bind simultaneously toat least two targets that are of different structure, e.g., twodifferent antigens, two different epitopes on the same antigen, or ahapten and/or an antigen or epitope. Multispecific, multivalentantibodies are constructs that have more than one binding site, and thebinding sites are of different specificity.

A bispecific antibody is an antibody that can bind simultaneously to twodifferent targets. Bispecific antibodies (bsAb) and bispecific antibodyfragments (bsFab) may have at least one arm that specifically binds to,for example, a tumor-associated antigen and at least one other arm thatspecifically binds to a targetable conjugate that bears a therapeutic ordiagnostic agent. A variety of bispecific fusion proteins can beproduced using molecular engineering.

PAM4 Antibody

Various embodiments of the invention concern antibodies that react withvery high selectivity with pancreatic cancer as opposed to normal orbenign pancreatic tissues. The anti-pancreatic cancer antibodies andfragments thereof are preferably raised against a crude mucinpreparation from a tumor of the human pancreas, although partiallypurified or even purified mucins may be utilized. A non-limiting exampleof such antibodies is the PAM4 antibody.

The murine PAM4 (mPAM4) antibody was developed by employing pancreaticcancer mucin derived from the xenografted RIP-1 human pancreaticcarcinoma as immunogen. (Gold et al., Int. J. Cancer, 57:204-210, 1994.)Antibody cross-reactivity and immunohistochemical staining studiesindicate that the PAM4 antibody recognizes a unique and novel epitope ona target pancreatic cancer antigen. Immunohistochemical stainingstudies, (see, e.g., U.S. Pat. No. 7,282,567), have shown that the PAM4MAb binds to an antigen expressed by breast, pancreas and other cancercells, with limited binding to normal human tissue; however, the highestexpression is usually by pancreatic cancer cells. Thus, the PAM4antibodies are relatively specific to pancreatic cancer andpreferentially bind pancreatic cancer cells. The PAM4 antibody isreactive with a target epitope which can be internalized. This epitopeis expressed primarily by antigens associated with pancreatic cancer andnot with focal pancreatitis or normal pancreatic tissue. Localizationand therapy studies using a radiolabeled PAM4 MAb in animal models havedemonstrated tumor targeting and therapeutic efficacy (Id.).

The PAM4 antibody exhibits several properties which make it a goodcandidate for clinical diagnostic and therapeutic applications. The PAM4antibody apparently recognizes an epitope of a pancreatic cancer antigenthat is distinct from the epitopes recognized by non-PAM4anti-pancreatic cancer antibodies (CA19.9, DUPAN2, SPAN1, Nd2, CEACAM5,CEACAM6, B72.3, anti-Le^(a), and other anti-Lewis antigens) (Id.).Antibodies suitable for use in combination or conjunction with PAM4antibody include, for example, CA19.9, DUPAN2, SPAN1, Nd2, B72.3, CC49,anti-CEACAM5, anti-CEACAM6, anti-Le, anti-HLA-DR, anti-CD40, anti-CD74,anti-CD138, and antibodies defined by the Lewis antigen Le(y), orantibodies against colon-specific antigen-p (CSAp), MUC-1, MUC-2, MUC-3,MUC-4, MUC-5ac, MUC-16, MUC-17, EGP-1, EGP-2, HER2/neu, EGFR,angiogenesis factors (e.g., VEGF and PlGF), insulin-like growth factor(ILGF), tenascin, platelet-derived growth factor, and IL-6, as well asproducts of oncogenes (bcl-2, Kras, p53), cMET, and antibodies againsttumor necrosis substances, such as described in patents by Epstein etal. (U.S. Pat. Nos. 6,071,491, 6,017,514, 5,019,368 and 5,882,626). Suchantibodies would be useful for complementing PAM4 antibody reactivitywith pancreatic cancer. These and other therapeutic agents could actsynergistically with PAM4 antibody, when administered before, togetherwith or after administration of PAM4 antibody.

Preferred embodiments may involve the use of a humanized PAM4 antibody.Because non-human monoclonal antibodies can be recognized by the humanhost as a foreign protein, and repeated injections can lead to harmfulhypersensitivity reactions, humanization of a murine antibody sequencescan reduce the adverse immune response that patients may experience. Formurine-based monoclonal antibodies, this is often referred to as a HumanAnti-Mouse Antibody (HAMA) response. Preferably some human residues inthe framework regions of the humanized PAM4 antibody or fragmentsthereof are replaced by their murine counterparts. It is also preferredthat a combination of framework sequences from two different humanantibodies is used for VH. The constant domains of the antibody moleculeare derived from those of a human antibody.

RS7 Antibody

The RS7 antibody is a murine IgG₁ raised against a crude membranepreparation of a human primary squamous cell lung carcinoma. (Stein etal., Cancer Res. 50: 1330, 1990) The RS7 antibody recognizes a 46-48 kDaglycoprotein, characterized as cluster 13. (Stein et al., Int. J. CancerSupp. 8:98-102, 1994) The antigen was designated as EGP-1 (epithelialglycoprotein-1), but is also referred to as Trop-2.

Trop-2 is a type-I transmembrane protein and has been cloned from bothhuman (Formaro et al., Int J Cancer 1995; 62:610-8) and mouse cells(Sewedy et al., Int J Cancer 1998; 75:324-30). In addition to its roleas a tumor-associated calcium signal transducer (Ripani et al., Int JCancer 1998; 76:671-6), the expression of human Trop-2 was shown to benecessary for tumorigenesis and invasiveness of colon cancer cells,which could be effectively reduced with a polyclonal antibody againstthe extracellular domain of Trop-2 (Wang et al., Mol Cancer Ther 2008;7:280-5).

The growing interest in Trop-2 as a therapeutic target for solid cancers(Cubas et al., Biochim Biophys Acta 2009; 1796:309-14) is attested byfurther reports that documented the clinical significance ofoverexpressed Trop-2 in breast (Huang et al., Clin Cancer Res 2005;11:4357-64), colorectal (Ohmachi et al., Clin Cancer Res 2006;12:3057-63; Fang et al., Int J Colorectal Dis 2009; 24:875-84), and oralsquamous cell (Fong et al., Modern Pathol 2008; 21:186-91) carcinomas.The latest evidence that prostate basal cells expressing high levels ofTrop-2 are enriched for in vitro and in vivo stem-like activity isparticularly noteworthy (Goldstein et al., Proc Natl Acad Sci USA 2008;105:20882-7).

Flow cytometry and immunohistochemical staining studies have shown thatthe RS7 MAb detects antigen on a variety of tumor types, with limitedbinding to normal human tissue. (Stein et al., 1990) EGP-1 is expressedprimarily by carcinomas such as carcinomas of the lung, stomach, urinarybladder, breast, ovary, uterus, and prostate. Localization and therapystudies using radiolabeled murine RS7 MAb in animal models havedemonstrated tumor targeting and therapeutic efficacy (Stein et al.,1990; Stein et al., 1991).

Strong RS7 staining has been demonstrated in tumors from the lung,breast, bladder, ovary, uterus, stomach, and prostate. (Stein et al.,Int. J. Cancer 55:938, 1993) The lung cancer cases comprised bothsquamous cell carcinomas and adenocarcinomas. (Id.) Both cell typesstained strongly, indicating that the RS7 antibody does not distinguishbetween histologic classes of non-small-cell carcinoma of the lung.

The RS7 MAb is rapidly internalized into target cells (Stein et al.,1993). The internalization rate constant for RS7 MAb is intermediatebetween the internalization rate constants of two other rapidlyinternalizing MAbs, which have been demonstrated to be useful forimmunotoxin production. (Id.) It is well documented that internalizationof immunotoxin conjugates is a requirement for anti-tumor activity.(Pastan et al., Cell 47:641, 1986) Internalization of drugimmunoconjugates has been described as a major factor in anti-tumorefficacy. (Yang et al., Proc. Nat'l Acad. Sci. USA 85: 1189, 1988) Thus,the RS7 antibody exhibits several important properties for therapeuticapplications.

Antibody Preparation

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A or Protein-G Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, seeBaines et al., “Purification of Immunoglobulin G (IgG),” in METHODS INMOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art, as discussedbelow.

Chimeric Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. General techniques for cloningmurine immunoglobulin variable domains are disclosed, for example, inOrlandi et al., Proc. Nat'l Acad. Sci. USA 6: 3833 (1989). Techniquesfor constructing chimeric antibodies are well known to those of skill inthe art. As an example, Leung et al., Hybridoma 13:469 (1994), producedan LL2 chimera by combining DNA sequences encoding the V_(κ) and V_(H)domains of murine LL2, an anti-CD22 monoclonal antibody, with respectivehuman V_(κ) and IgG₁ constant region domains.

Humanized Antibodies

Techniques for producing humanized MAbs are well known in the art (see,e.g., Jones et al., Nature 321: 522 (1986), Riechmann et al., Nature332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988), Carter etal., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev.Biotech. 12: 437 (1992), and Singer et al., J. Immun. 150: 2844 (1993)).A chimeric or murine monoclonal antibody may be humanized bytransferring the mouse CDRs from the heavy and light variable chains ofthe mouse immunoglobulin into the corresponding variable domains of ahuman antibody. The mouse framework regions (FR) in the chimericmonoclonal antibody are also replaced with human FR sequences. As simplytransferring mouse CDRs into human FRs often results in a reduction oreven loss of antibody affinity, additional modification might berequired in order to restore the original affinity of the murineantibody. This can be accomplished by the replacement of one or morehuman residues in the FR regions with their murine counterparts toobtain an antibody that possesses good binding affinity to its epitope.See, for example, Tempest et al., Biotechnology 9:266 (1991) andVerhoeyen et al., Science 239: 1534 (1988). Preferred residues forsubstitution include FR residues that are located within 1, 2, or 3Angstroms of a CDR residue side chain, that are located adjacent to aCDR sequence, or that are predicted to interact with a CDR residue.

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Pharmacol.3:544-50). A fully human antibody also can be constructed by genetic orchromosomal transfection methods, as well as phage display technology,all of which are known in the art. See for example, McCafferty et al.,Nature 348:552-553 (1990). Such fully human antibodies are expected toexhibit even fewer side effects than chimeric or humanized antibodiesand to function in vivo as essentially endogenous human antibodies.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40). Human antibodies may be generated from normal humans or fromhumans that exhibit a particular disease state, such as cancer(Dantas-Barbosa et al., 2005). The advantage to constructing humanantibodies from a diseased individual is that the circulating antibodyrepertoire may be biased towards antibodies against disease-associatedantigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.). Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.). RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97).Library construction was performed according to Andris-Widhopf et al.(2000, In: Phage Display Laboratory Manual, Barbas et al. (eds), 1^(st)edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.pp. 9.1 to 9.22). The final Fab fragments were digested with restrictionendonucleases and inserted into the bacteriophage genome to make thephage display library. Such libraries may be screened by standard phagedisplay methods, as known in the art. Phage display can be performed ina variety of formats, for their review, see e.g. Johnson and Chiswell,Current Opinion in Structural Biology 3:5564-571 (1993).

Human antibodies may also be generated by in vitro activated B-cells.See U.S. Pat. Nos. 5,567,610 and 5,229,275, incorporated herein byreference in their entirety. The skilled artisan will realize that thesetechniques are exemplary and any known method for making and screeninghuman antibodies or antibody fragments may be utilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols. Methods for obtaining human antibodies fromtransgenic mice are disclosed by Green et al., Nature Genet. 7:13(1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int.Immun. 6:579 (1994). A non-limiting example of such a system is theXenoMouse® (e.g., Green et al., 1999, J. Immunol. Methods 231:11-23,incorporated herein by reference) from Abgenix (Fremont, Calif.). In theXenoMouse® and similar animals, the mouse antibody genes have beeninactivated and replaced by functional human antibody genes, while theremainder of the mouse immune system remains intact.

The XenoMouse® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH andIgkappa loci, including the majority of the variable region sequences,along with accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B-cells,which may be processed into hybridomas by known techniques. A XenoMouse®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XenoMouse®are available, each of which is capable of producing a different classof antibody. Transgenically produced human antibodies have been shown tohave therapeutic potential, while retaining the pharmacokineticproperties of normal human antibodies (Green et al., 1999). The skilledartisan will realize that the claimed compositions and methods are notlimited to use of the XenoMouse® system but may utilize any transgenicanimal that has been genetically engineered to produce human antibodies.

Known Antibodies and Target Antigens

As discussed above, in preferred embodiments the immunoconjugateantibodies are of use for treatment of pancreatic cancer. However, theskilled artisan will realize that the invention is not so limited andmay be applied to other types of cancer or even other disease states.Non-limiting examples include malignant disease, cardiovascular disease,infectious disease, inflammatory disease, autoimmune disease, immunedysfunction disease (e.g., graft versus host disease or organ transplantrejection) or neurological disease. Exemplary target antigens of use fortreating such diseases may include carbonic anhydrase IX, CCCL19,CCCL21, CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15,CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD29, CD30,CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55,CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126,CD133, CD138, CD147, CD154, CXCR4, AFP, PSMA, CEACAM5, CEACAM6, B7, ED-Bof fibronectin, Factor H, FHL-1, Flt-3, folate receptor, GROB, HMGB-1,hypoxia inducible factor (HIF), HM1.24, insulin-like growth factor-1(ILGF-1), IFN-γ, IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R,IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10,MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5,NCA-95, NCA-90, Ia, HM1.24, EGP-1, EGP-2, HLA-DR, tenascin, Le(y),RANTES, T101, TAC, Tn antigen, Thomson-Friedenreich antigens, tumornecrosis antigens, TNF-α, TRAIL receptor (R1 and R2), VEGFR, EGFR, PlGF,complement factors C3, C3a, C3b, C5a, C5, and an oncogene product.

In certain embodiments, such as treating tumors, antibodies of use maytarget tumor-associated antigens. These antigenic markers may besubstances produced by a tumor or may be substances which accumulate ata tumor site, on tumor cell surfaces or within tumor cells. Among suchtumor-associated markers are those disclosed by Herberman,“Immunodiagnosis of Cancer”, in Fleisher ed., “The Clinical Biochemistryof Cancer”, page 347 (American Association of Clinical Chemists, 1979)and in U.S. Pat. Nos. 4,150,149; 4,361,544; and 4,444,744, the Examplessection of each of which is incorporated herein by reference. Reports ontumor associated antigens (TAAs) include Mizukami et al., (2005, NatureMed. 11:992-97); Hatfield et al., (2005, Curr. Cancer Drug Targets5:229-48); Vallbohmer et al. (2005, J. Clin. Oncol. 23:3536-44); and Renet al. (2005, Ann. Surg. 242:55-63), each incorporated herein byreference with respect to the TAAs identified.

Tumor-associated markers have been categorized by Herberman, supra, in anumber of categories including oncofetal antigens, placental antigens,oncogenic or tumor virus associated antigens, tissue associatedantigens, organ associated antigens, ectopic hormones and normalantigens or variants thereof. Occasionally, a sub-unit of atumor-associated marker is advantageously used to raise antibodieshaving higher tumor-specificity, e.g., the beta-subunit of humanchorionic gonadotropin (HCG) or the gamma region of carcinoembryonicantigen (CEA), which stimulate the production of antibodies having agreatly reduced cross-reactivity to non-tumor substances as disclosed inU.S. Pat. Nos. 4,361,644 and 4,444,744.

Another marker of interest is transmembrane activator andCAML-interactor (TACI). See Yu et al. Nat. Immunol. 1:252-256 (2000).Briefly, TACI is a marker for B-cell malignancies (e.g., lymphoma). TACIand B cell maturation antigen (BCMA) are bound by the tumor necrosisfactor homolog—a proliferation-inducing ligand (APRIL). APRIL stimulatesin vitro proliferation of primary B and T cells and increases spleenweight due to accumulation of B cells in vivo. APRIL also competes withTALL-I (also called BLyS or BAFF) for receptor binding. Soluble BCMA andTACI specifically prevent binding of APRIL and block APRIL-stimulatedproliferation of primary B cells. BCMA-Fc also inhibits production ofantibodies against keyhole limpet hemocyanin and Pneumovax in mice,indicating that APRIL and/or TALL-I signaling via BCMA and/or TACI arerequired for generation of humoral immunity. Thus, APRIL-TALL-I andBCMA-TACI form a two ligand-two receptor pathway involved in stimulationof B and T cell function.

Where the disease involves a lymphoma, leukemia or autoimmune disorder,targeted antigens may be selected from the group consisting of CD4, CD5,CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38,CD40, CD40L, CD46, CD52, CD54, CD67, CD74, CD79a, CD80, CD126, CD138,CD154, B7, MUC1, Ia, Ii, HM1.24, HLA-DR, tenascin, VEGF, PlGF, ED-Bfibronectin, an oncogene, an oncogene product, CD66a-d, necrosisantigens, IL-2, T101, TAG, IL-6, MIF, TRAIL-R1 (DR4) and TRAIL-R2 (DR5).

The skilled artisan will realize that any antibody or fragment known inthe art that has binding specificity for a target antigen associatedwith a disease state or condition may be utilized. Such known antibodiesinclude, but are not limited to, hR1 (anti-IGF-1R, U.S. patentapplication Ser. No. 12/772,645, filed Mar. 12, 2010) hPAM4(anti-pancreatic cancer mucin, U.S. Pat. No. 7,282,567), hA20(anti-CD20, U.S. Pat. No. 7,251,164), hA19 (anti-CD19, U.S. Pat. No.7,109,304), hIMMU31 (anti-AFP, U.S. Pat. No. 7,300,655), hLL1(anti-CD74, U.S. Pat. No. 7,312,318), hLL2 (anti-CD22, U.S. Pat. No.7,074,403), hMu-9 (anti-CSAp, U.S. Pat. No. 7,387,773), hL243(anti-HLA-DR, U.S. Pat. No. 7,612,180), hMN-14 (anti-CEACAM5, U.S. Pat.No. 6,676,924), hMN-15 (anti-CEACAM6, U.S. Pat. No. 7,662,378, U.S.patent application Ser. No. 12/846,062, filed Jul. 29, 2010), hRS7(anti-EGP-1, U.S. Pat. No. 7,238,785), hMN-3 (anti-CEACAM6, U.S. Pat.No. 7,541,440), Ab124 and Ab125 (anti-CXCR4, U.S. Pat. No. 7,138,496)the Examples section of each cited patent or application incorporatedherein by reference.

Various other antibodies of use are known in the art (e.g., U.S. Pat.Nos. 5,686,072; 5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104;6,730.300; 6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084;7,238,785; 7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318;7,585,491; 7,612,180; 7,642,239 and U.S. Patent Application Publ. No.20060193865; each incorporated herein by reference.)

Antibodies of use may be commercially obtained from a wide variety ofknown sources. For example, a variety of antibody secreting hybridomalines are available from the American Type Culture Collection (ATCC,Manassas, Va.). A large number of antibodies against various diseasetargets, including but not limited to tumor-associated antigens, havebeen deposited at the ATCC and/or have published variable regionsequences and are available for use in the claimed methods andcompositions. See, e.g., U.S. Pat. Nos. 7,312,318; 7,282,567; 7,151,164;7,074,403; 7,060,802; 7,056,509; 7,049,060; 7,045,132; 7,041,803;7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598;6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018;6,964,854; 6,962,981; 6,962,813; 6,956,107; 6,951,924; 6,949,244;6,946,129; 6,943,020; 6,939,547; 6,921,645; 6,921,645; 6,921,533;6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625;6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580;6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226;6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778; 6,812,206;6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681;6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,155; 6,716,966;6,709,653; 6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355;6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852;6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279;6,596,852; 6,592,868; 6,576,745; 6,572,856; 6,566,076; 6,562,618;6,545,130; 6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227;6,518,404; 6,511,665; 6,491,915; 6,488,930; 6,482,598; 6,482,408;6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356;6,455,044; 6,455,040, 6,451,310; 6,444,206, 6,441,143; 6,432,404;6,432,402; 6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091;6,395,276; 6,395,274; 6,387,350; 6,383,759; 6,383,484; 6,376,654;6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244;6,346,246; 6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393;6,254,868; 6,187,287; 6,183,744; 6,129,914; 6,120,767; 6,096,289;6,077,499; 5,922,302; 5,874,540; 5,814,440; 5,798,229; 5,789,554;5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953,5,525,338. These are exemplary only and a wide variety of otherantibodies and their hybridomas are known in the art. The skilledartisan will realize that antibody sequences or antibody-secretinghybridomas against almost any disease-associated antigen may be obtainedby a simple search of the ATCC, NCBI and/or USPTO databases forantibodies against a selected disease-associated target of interest. Theantigen binding domains of the cloned antibodies may be amplified,excised, ligated into an expression vector, transfected into an adaptedhost cell and used for protein production, using standard techniqueswell known in the art.

Antibody Fragments

Antibody fragments are antigen binding portions of an antibody, such asF(ab′)₂, Fab′, F(ab)₂, Fab, Fv, sFv, scFv and the like. Antibodyfragments which recognize specific epitopes can be generated by knowntechniques. F(ab′)₂ fragments, for example, can be produced by pepsindigestion of the antibody molecule. These and other methods aredescribed, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647 and references contained therein. Also, see Nisonoff et al.,Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119(1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page 422(Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4. Alternatively, Fab′ expression libraries can beconstructed (Huse et al., 1989, Science, 246:1274-1281) to allow rapidand easy identification of monoclonal Fab′ fragments with the desiredspecificity.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). A scFvmolecule is denoted as either VL-L-VH if the VL domain is the N-terminalpart of the scFv molecule, or as VH-L-VL if the VH domain is theN-terminal part of the scFv molecule. Methods for making scFv moleculesand designing suitable peptide linkers are described in U.S. Pat. No.4,704,692, U.S. Pat. No. 4,946,778, R. Raag and M. Whitlow, “SingleChain Fvs.” FASEB Vol 9:73-80 (1995) and R. E. Bird and B. W. Walker,Single Chain Antibody Variable Regions, TIBTECH, Vol 9: 132-137 (1991).

Other antibody fragments, for example single domain antibody fragments,are known in the art and may be used in the claimed constructs. Singledomain antibodies (VHH) may be obtained, for example, from camels,alpacas or llamas by standard immunization techniques. (See, e.g.,Muyldermans et al., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods281:161-75, 2003; Maass et al., J Immunol Methods 324:13-25, 2007). TheVHH may have potent antigen-binding capacity and can interact with novelepitopes that are inaccessible to conventional VH-VL pairs. (Muyldermanset al., 2001). Alpaca serum IgG contains about 50% camelid heavy chainonly IgG antibodies (HCAbs) (Maass et al., 2007). Alpacas may beimmunized with known antigens, such as TNF-α, and VHHs can be isolatedthat bind to and neutralize the target antigen (Maass et al., 2007). PCRprimers that amplify virtually all alpaca VHH coding sequences have beenidentified and may be used to construct alpaca VHH phage displaylibraries, which can be used for antibody fragment isolation by standardbiopanning techniques well known in the art (Maass et al., 2007).

An antibody fragment can also be prepared by proteolytic hydrolysis of afull-length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full-length antibodies by conventionalmethods. For example, an antibody fragment can be produced by enzymaticcleavage of antibodies with pepsin to provide an approximate 100 kDfragment denoted F(ab′)₂. This fragment can be further cleaved using athiol reducing agent, and optionally a blocking group for the sulfhydrylgroups resulting from cleavage of disulfide linkages, to produce anapproximate 50 Kd Fab′ monovalent fragment. Alternatively, an enzymaticcleavage using papain produces two monovalent Fab fragments and an Fcfragment directly.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

General Techniques for Antibody Cloning and Production

Various techniques, such as production of chimeric or humanizedantibodies, may involve procedures of antibody cloning and construction.The antigen-binding V_(κ) (variable light chain) and V_(H) (variableheavy chain) sequences for an antibody of interest may be obtained by avariety of molecular cloning procedures, such as RT-PCR, 5′-RACE, andcDNA library screening. The V genes of a MAb from a cell that expressesa murine MAb can be cloned by PCR amplification and sequenced. Toconfirm their authenticity, the cloned V_(L) and V_(H) genes can beexpressed in cell culture as a chimeric Ab as described by Orlandi etal., (Proc. Natl. Acad. Sci., USA, 86: 3833 (1989)). Based on the V genesequences, a humanized MAb can then be designed and constructed asdescribed by Leung et al. (Mol. Immunol., 32: 1413 (1995)).

cDNA can be prepared from any known hybridoma line or transfected cellline producing a murine MAb by general molecular cloning techniques(Sambrook et al., Molecular Cloning, A laboratory manual, 2^(nd) Ed(1989)). The Vκ sequence for the MAb may be amplified using the primersVK1BACK and VK1FOR (Orlandi et al., 1989) or the extended primer setdescribed by Leung et al. (BioTechniques, 15: 286 (1993)). The V_(H)sequences can be amplified using the primer pair VH1BACK/VH1FOR (Orlandiet al., 1989) or the primers annealing to the constant region of murineIgG described by Leung et al. (Hybridoma, 13:469 (1994)). Humanized Vgenes can be constructed by a combination of long oligonucleotidetemplate syntheses and PCR amplification as described by Leung et al.(Mol. Immunol., 32: 1413 (1995)).

PCR products for Vκ can be subcloned into a staging vector, such as apBR327-based staging vector, VKpBR, that contains an Ig promoter, asignal peptide sequence and convenient restriction sites. PCR productsfor V_(H) can be subcloned into a similar staging vector, such as thepBluescript-based VHpBS. Expression cassettes containing the Vκ andV_(H) sequences together with the promoter and signal peptide sequencescan be excised from VKpBR and VHpBS and ligated into appropriateexpression vectors, such as pKh and pG1g, respectively (Leung et al.,Hybridoma, 13:469 (1994)). The expression vectors can be co-transfectedinto an appropriate cell and supernatant fluids monitored for productionof a chimeric, humanized or human MAb. Alternatively, the Vκ and V_(H)expression cassettes can be excised and subcloned into a singleexpression vector, such as pdHL2, as described by Gillies et al. (J.Immunol. Methods 125:191 (1989) and also shown in Losman et al., Cancer,80:2660 (1997)).

In an alternative embodiment, expression vectors may be transfected intohost cells that have been pre-adapted for transfection, growth andexpression in serum-free medium. Exemplary cell lines that may be usedinclude the Sp/EEE, Sp/ESF and Sp/ESF-X cell lines (see, e.g., U.S. Pat.Nos. 7,531,327; 7,537,930 and 7,608,425; the Examples section of each ofwhich is incorporated herein by reference). These exemplary cell linesare based on the Sp2/0 myeloma cell line, transfected with a mutantBcl-EEE gene, exposed to methotrexate to amplify transfected genesequences and pre-adapted to serum-free cell line for proteinexpression.

Bispecific and Multispecific Antibodies

As discussed in the Examples below, in preferred embodiments theradiolabeled antibody and the drug-conjugated antibody are administeredas separate antibodies, either sequentially or concurrently. However, inalternative embodiments the two immunoconjugates may be administered asa single bispecific or multispecific antibody. Numerous methods toproduce bispecific or multispecific antibodies are known, as disclosed,for example, in U.S. Pat. No. 7,405,320, the Examples section of whichis incorporated herein by reference. Bispecific antibodies can beproduced by the quadroma method, which involves the fusion of twodifferent hybridomas, each producing a monoclonal antibody recognizing adifferent antigenic site (Milstein and Cuello, Nature, 1983;305:537-540).

Another method for producing bispecific antibodies usesheterobifunctional cross-linkers to chemically tether two differentmonoclonal antibodies (Staerz, et al. Nature. 1985; 314:628-631; Perez,et al. Nature. 1985; 316:354-356). Bispecific antibodies can also beproduced by reduction of each of two parental monoclonal antibodies tothe respective half molecules, which are then mixed and allowed toreoxidize to obtain the hybrid structure (Staerz and Bevan. Proc NatlAcad Sci USA. 1986; 83:1453-1457). Other methods include improving theefficiency of generating hybrid hybridomas by gene transfer of distinctselectable markers via retrovirus-derived shuttle vectors intorespective parental hybridomas, which are fused subsequently (DeMonte,et al. Proc Natl Acad Sci USA. 1990, 87:2941-2945); or transfection of ahybridoma cell line with expression plasmids containing the heavy andlight chain genes of a different antibody.

Cognate V_(H) and V_(L) domains can be joined with a peptide linker ofappropriate composition and length (usually consisting of more than 12amino acid residues) to form a single-chain Fv (scFv), as discussedabove. Reduction of the peptide linker length to less than 12 amino acidresidues prevents pairing of V_(H) and V_(L) domains on the same chainand forces pairing of V_(H) and V_(L) domains with complementary domainson other chains, resulting in the formation of functional multimers.Polypeptide chains of V_(H) and V_(L) domains that are joined withlinkers between 3 and 12 amino acid residues form predominantly dimers(termed diabodies). With linkers between 0 and 2 amino acid residues,trimers (termed triabody) and tetramers (termed tetrabody) are favored,but the exact patterns of oligomerization appear to depend on thecomposition as well as the orientation of V-domains (V_(H)-linker-V_(L)or V_(L)-linker-V_(H)), in addition to the linker length.

These techniques for producing multispecific or bispecific antibodiesexhibit various difficulties in terms of low yield, necessity forpurification, low stability or the labor-intensiveness of the technique.More recently, a technique known as “dock and lock” (DNL), discussed inmore detail below, has been utilized to produce combinations ofvirtually any desired antibodies, antibody fragments and other effectormolecules. Any of the techniques known in the art for making bispecificor multispecific antibodies may be utilized in the practice of thepresently claimed methods.

Dock-and-Lock (DNL)

Bispecific or multispecific antibodies or other constructs may beproduced using the dock-and-lock technology (see, e.g., U.S. Pat. Nos.7,550,143; 7,521,056; 7,534,866; 7,527,787 and 7,666,400, the Examplessection of each incorporated herein by reference). The DNL methodexploits specific protein/protein interactions that occur between theregulatory (R) subunits of cAMP-dependent protein kinase (PKA) and theanchoring domain (AD) of A-kinase anchoring proteins (AKAPs) (Baillie etal., FEBS Letters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol. Cell.Biol. 2004; 5: 959). PKA, which plays a central role in one of the beststudied signal transduction pathways triggered by the binding of thesecond messenger cAMP to the R subunits, was first isolated from rabbitskeletal muscle in 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763).The structure of the holoenzyme consists of two catalytic subunits heldin an inactive form by the R subunits (Taylor, J. Biol. Chem. 1989;264:8443). Isozymes of PKA are found with two types of R subunits (RIand RII), and each type has α and β isoforms (Scott, Pharmacol. Ther.1991; 50:123). The R subunits have been isolated only as stable dimersand the dimerization domain has been shown to consist of the first 44amino-terminal residues (Newlon et al., Nat. Struct. Biol. 1999; 6:222).Binding of cAMP to the R subunits leads to the release of activecatalytic subunits for a broad spectrum of serine/threonine kinaseactivities, which are oriented toward selected substrates through thecompartmentalization of PKA via its docking with AKAPs (Scott et al., J.Biol. Chem. 1990; 265; 21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci. USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell. Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for R11dimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). AKAPs will only bind to dimeric R subunits. For humanRIIα, the AD binds to a hydrophobic surface formed by the 23amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;6:216). Thus, the dimerization domain and AKAP binding domain of humanRIIα are both located within the same N-terminal 44 amino acid sequence(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J.2001; 20:1651), which is termed the DDD herein.

We have developed a platfoiin technology to utilize the DDD of humanRIIα and the AD of AKAP as an excellent pair of linker modules fordocking any two entities, referred to hereafter as A and B, into anoncovalent complex, which could be further locked into a stablytethered structure through the introduction of cysteine residues intoboth the DDD and AD at strategic positions to facilitate the formationof disulfide bonds. The general methodology of the “dock-and-lock”approach is as follows. Entity A is constructed by linking a DDDsequence to a precursor of A, resulting in a first component hereafterreferred to as a. Because the DDD sequence would effect the spontaneousformation of a dimer, A would thus be composed of a₂. Entity B isconstructed by linking an AD sequence to a precursor of B, resulting ina second component hereafter referred to as b. The dimeric motif of DDDcontained in a₂ will create a docking site for binding to the ADsequence contained in b, thus facilitating a ready association of a₂ andb to form a binary, trimeric complex composed of a₂b. This binding eventis made irreversible with a subsequent reaction to covalently secure thetwo entities via disulfide bridges, which occurs very efficiently basedon the principle of effective local concentration because the initialbinding interactions should bring the reactive thiol groups placed ontoboth the DDD and AD into proximity (Chmura et al., Proc. Natl. Acad.Sci. USA. 2001; 98:8480) to ligate site-specifically. Using variouscombinations of linkers, adaptor modules and precursors, a wide varietyof DNL constructs of different stoichiometry may be produced and used,including but not limited to dimeric, trimeric, tetrameric, pentamericand hexameric DNL constructs (see, e.g., U.S. Pat. Nos. 7,550,143;7,521,056; 7,534,866; 7,527,787 and 7,666,400.)

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors described in the Examples below,virtually any protein or peptide may be incorporated into a DNLconstruct. However, the technique is not limiting and other methods ofconjugation may be utilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the part(s) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

Pre-Targeting

In preferred embodiments, the radionuclide and drug are directlyattached to the antibodies of interest and administered asimmunoconjugates. However, in alternative embodiments the radionuclideand/or drug may be conjugated to a targetable construct that comprisesone or more haptens. The hapten is recognized by at least one arm of abispecific or multispecific antibody that also binds to atumor-associated antigen or other disease-associated antigen. In thiscase, the radionuclide and/or drug bind indirectly to the antibodies,via the binding of the targetable construct. This process is referred toas pretargeting.

Pre-targeting is a multistep process originally developed to resolve theslow blood clearance of directly targeting antibodies, which contributesto undesirable toxicity to normal tissues such as bone marrow. Withpre-targeting, a radionuclide or other diagnostic or therapeutic agentis attached to a small delivery molecule (targetable construct) that iscleared within minutes from the blood. A pre-targeting bispecific ormultispecific antibody, which has binding sites for the targetableconstruct as well as a target antigen, is administered first, freeantibody is allowed to clear from circulation and then the targetableconstruct is administered.

Pre-targeting methods are disclosed, for example, in Goodwin et al.,U.S. Pat. No. 4,863,713; Goodwin et al., J. Nucl. Med. 29:226, 1988;Hnatowich et al., J. Nucl. Med. 28:1294, 1987; Oehr et al., J. Nucl.Med. 29:728, 1988; Klibanov et al., J. Nucl. Med. 29:1951, 1988;Sinitsyn et al., J. Nucl. Med. 30:66, 1989; Kalofonos et al., J. Nucl.Med. 31:1791, 1990; Schechter et al., Int. J. Cancer 48:167, 1991;Paganelli et al., Cancer Res. 51:5960, 1991; Paganelli et al., Nucl.Med. Commun. 12:211, 1991; U.S. Pat. No. 5,256,395; Stickney et al.,Cancer Res. 51:6650, 1991; Yuan et al., Cancer Res. 51:3119, 1991; U.S.Pat. Nos. 6,077,499; 7,011,812; 7,300,644; 7,074,405; 6,962,702;7,387,772; 7,052,872; 7,138,103; 6,090,381; 6,472,511; 6,962,702; and6,962,702, each incorporated herein by reference.

A pre-targeting method of treating or diagnosing a disease or disorderin a subject may be provided by: (1) administering to the subject abispecific antibody or antibody fragment; (2) optionally administeringto the subject a clearing composition, and allowing the composition toclear the antibody from circulation; and (3) administering to thesubject the targetable construct, containing one or more chelated orchemically bound therapeutic or diagnostic agents.

Immunoconjugates

In preferred embodiments, the radionuclide and drug are attacheddirectly to an antibody or antibody fragment to form an immunoconjugate.Immunoconjugates comprising radionuclides may be formed by directcovalent attachment of the radionuclide to a functional group on theantibody, as in the well known radioiodination of tyrosine residues.

Alternatively, the radionuclide may be conjugated to a chelating moietythat is attached to the antibody or fragment thereof. Methods forcovalent conjugation of chelating moieties, drugs and other therapeuticagents to antibodies and other proteins are known in the art and anysuch known method may be utilized.

Therapeutic agents may be attached, for example to reduced SH groupsand/or to carbohydrate side chains. A therapeutic agent can be attachedat the hinge region of a reduced antibody component via disulfide bondformation. Alternatively, such agents can be attached using aheterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)propionate (SPDP). Yu et al., Int. J. Cancer 56: 244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION ANDCROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLESAND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). Alternatively, the therapeutic agentcan be conjugated via a carbohydrate moiety in the Fc region of theantibody. The carbohydrate group can be used to increase the loading ofthe same agent that is bound to a thiol group, or the carbohydratemoiety can be used to bind a different therapeutic or diagnostic agent.

Methods for conjugating functional groups to antibodies via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, the Examples section of which is incorporated herein byreference. The general method involves reacting an antibody having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function. This reaction results in an initial Schiff base(imine) linkage, which can be stabilized by reduction to a secondaryamine to form the final conjugate.

The Fc region may be absent if the antibody component of theimmunoconjugate is an antibody fragment. However, it is possible tointroduce a carbohydrate moiety into the light chain variable region ofa full length antibody or antibody fragment. See, for example, Leung etal., J. Immunol. 154: 5919 (1995); U.S. Pat. Nos. 5,443,953 and6,254,868, the Examples section of which is incorporated herein byreference. The engineered carbohydrate moiety is used to attach thetherapeutic or diagnostic agent.

Methods of conjugation and use of chelating agents to attachradionuclides to proteins are well known in the art (see, e.g., U.S.Pat. No. 7,563,433, the Examples section of which is incorporated hereinby reference). Exemplary chelators include but are not limited to DTPA(such as Mx-DTPA), DOTA, TETA, NETA or NOTA. Chelates may be directlylinked to antibodies or peptides, for example as disclosed in U.S. Pat.No. 4,824,659, incorporated herein in its entirety by reference.Particularly useful chelating moieties include 2-benzyl-DTPA and itsmonomethyl and cyclohexyl analogs. Other ring-type chelates such asmacrocyclic polyethers are of interest for stably binding radionuclides.

In certain embodiments, radionuclides may be attached to proteins orpeptides by reaction with a reagent having a long tail, to which may beattached a multiplicity of chelating groups for binding ions. Such atail can be a polymer such as a polylysine, polysaccharide, or otherderivatized or derivatizable chains having pendant groups to which canbe bound chelating groups such as, e.g., ethylenediaminetetraacetic acid(EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins,polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and likegroups known to be useful for this purpose.

An alternative method for attaching chelating moieties, drugs or otherfunctional groups to a targeting molecule involves use of clickchemistry reactions. The click chemistry approach was originallyconceived as a method to rapidly generate complex substances by joiningsmall subunits together in a modular fashion. (See, e.g., Kolb et al.,2004, Angew Chem Int Ed 40:3004-31; Evans, 2007, Aust J Chem 60:384-95.)Various forms of click chemistry reaction are known in the art, such asthe Huisgen 1,3-dipolar cycloaddition copper catalyzed reaction (Tornoeet al., 2002, J Organic Chem 67:3057-64), which is often referred to asthe “click reaction.” Other alternatives include cycloaddition reactionssuch as the Diels-Alder, nucleophilic substitution reactions (especiallyto small strained rings like epoxy and aziridine compounds), carbonylchemistry formation of urea compounds and reactions involvingcarbon-carbon double bonds, such as alkynes in thiol-yne reactions.

The azide alkyne Huisgen cycloaddition reaction uses a copper catalystin the presence of a reducing agent to catalyze the reaction of aterminal alkyne group attached to a first molecule. In the presence of asecond molecule comprising an azide moiety, the azide reacts with theactivated alkyne to form a 1,4-disubstituted 1,2,3-triazole. The coppercatalyzed reaction occurs at room temperature and is sufficientlyspecific that purification of the reaction product is often notrequired. (Rostovstev et al., 2002, Angew Chem Int Ed 41:2596; Tornoe etal., 2002, J Org Chem 67:3057.) The azide and alkyne functional groupsare largely inert towards biomolecules in aqueous medium, allowing thereaction to occur in complex solutions. The triazole formed ischemically stable and is not subject to enzymatic cleavage, making theclick chemistry product highly stable in biological systems. Althoughthe copper catalyst is toxic to living cells, the copper-based clickchemistry reaction may be used in vitro for immunoconjugate formation.

A copper-free click reaction has been proposed for covalent modificationof biomolecules. (See, e.g., Agard et al., 2004, J Am Chem Soc126:15046-47.) The copper-free reaction uses ring strain in place of thecopper catalyst to promote a [3+2] azide-alkyne cycloaddition reaction(Id.) For example, cyclooctyne is an 8-carbon ring structure comprisingan internal alkyne bond. The closed ring structure induces a substantialbond angle deformation of the acetylene, which is highly reactive withazide groups to form a triazole. Thus, cyclooctyne derivatives may beused for copper-free click reactions (Id.)

Another type of copper-free click reaction was reported by Ning et al.(2010, Angew Chem Int Ed 49:3065-68), involving strain-promotedalkyne-nitrone cycloaddition. To address the slow rate of the originalcyclooctyne reaction, electron-withdrawing groups are attached adjacentto the triple bond (Id.) Examples of such substituted cyclooctynesinclude difluorinated cyclooctynes, 4-dibenzocyclooctynol andazacyclooctyne (Id.) An alternative copper-free reaction involvedstrain-promoted akyne-nitrone cycloaddition to give N-alkylatedisoxazolines (Id.) The reaction was reported to have exceptionally fastreaction kinetics and was used in a one-pot three-step protocol forsite-specific modification of peptides and proteins (Id.) Nitrones wereprepared by the condensation of appropriate aldehydes withN-methylhydroxylamine and the cycloaddition reaction took place in amixture of acetonitrile and water (Id.) These and other known clickchemistry reactions may be used to attach chelating moieties or drugs toantibodies in vitro.

Methods of Therapeutic Treatment

Various embodiments concern methods of treating a cancer in a subject,comprising administering a therapeutically effective amount of anantibody, fragment or immunoconjugate. In preferred embodiments, thesubject is administered a radionuclide-conjugated antibody or fragmentand a drug-conjugated antibody or fragment.

The immunoconjugates can be supplemented with the administration, eitherconcurrently or sequentially, of at least one other therapeutic agent.Multimodal therapies may include therapy with other antibodies, such asanti-CD22, anti-CD19, anti-CD20, anti-CD21, anti-CD74, anti-CD80,anti-CD23, anti-CD45, anti-CD46, anti-MIF, anti-EGP-1, anti-CEACAM5,anti-CEACAM6, anti-pancreatic cancer mucin, anti-IGF-1R or anti-HLA-DR(including the invariant chain) antibodies in the form of nakedantibodies, fusion proteins, or as immunoconjugates. Various antibodiesof use, such as anti-CD19, anti-CD20, and anti-CD22 antibodies, areknown to those of skill in the art. See, for example, Ghetie et al.,Cancer Res. 48:2610 (1988); Hekman et al., Cancer Immunol. Immunother.32:364 (1991); Longo, Curr. Opin. Oncol. 8:353 (1996), U.S. Pat. Nos.5,798,554; 6,187,287; 6,306,393; 6,676,924; 7,109,304; 7,151,164;7,230,084; 7,230,085; 7,238,785; 7,238,786; 7,282,567; 7,300,655;7,312,318; 7,612,180; 7,501,498; the Examples section of each of whichis incorporated herein by reference.

In one non-limiting embodiment, the present invention contemplatestreatment with conjugated PAM4 and RS7 antibodies or fragments thereofbefore, in combination with, or after other pancreatic tumor associatedantibodies such as CA19.9, DUPAN2, SPAN1, Nd2, B72.3, CC49, anti-Le^(a)antibodies, and antibodies to other Lewis antigens (e.g., Le(y)), aswell as antibodies against carcinoembryonic antigen (CEA or CEACAM5),CEACAM6, colon-specific antigen-p (CSAp), MUC-1, MUC-2, MUC-3, MUC-4,MUC-5ac, MUC-16, MUC-17, HLA-DR, CD40, CD74, CD138, HER2/neu, EGFR,EGP-1, EGP-2, angiogenesis factors (e.g., VEGF, PlGF), insulin-likegrowth factor (ILGF), tenascin, platelet-derived growth factor, andIL-6, as well as products of oncogenes (e.g., bcl-2, Kras, p53), cMET,and antibodies against tumor necrosis substances. These solid tumorantibodies may be naked or conjugated to, inter alia, drugs, toxins,isotopes, radionuclides or immunomodulators. Many different antibodycombinations may be constructed and used in either naked or conjugatedform. Alternatively, different antibody combinations may be employed foradministration in combination with other therapeutic agents, such as acytotoxic drug or with radiation, given consecutively, simultaneously,or sequentially.

In another form of multimodal therapy, subjects receive immunoconjugatesin conjunction with standard cancer chemotherapy. For example, “CVB”(1.5 g/m² cyclophosphamide, 200-400 mg/m² etoposide, and 150-200 mg/m²carmustine) is a regimen used to treat non-Hodgkin's lymphoma. Patti etal., Eur. J. Haematol. 51: 18 (1993). Other suitable combinationchemotherapeutic regimens are well-known to those of skill in the art.See, for example, Freedman et al., “Non-Hodgkin's Lymphomas,” in CANCERMEDICINE, VOLUME 2, 3rd Edition, Holland et al. (eds.), pages 2028-2068(Lea & Febiger 1993). As an illustration, first generationchemotherapeutic regimens for treatment of intermediate-gradenon-Hodgkin's lymphoma (NHL) include C-MOPP (cyclophosphamide,vincristine, procarbazine and prednisone) and CHOP (cyclophosphamide,doxorubicin, vincristine, and prednisone). A useful second generationchemotherapeutic regimen is m-BACOD (methotrexate, bleomycin,doxorubicin, cyclophosphamide, vincristine, dexamethasone andleucovorin), while a suitable third generation regimen is MACOP-B(methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone,bleomycin and leucovorin). Additional useful drugs include phenylbutyrate, bendamustine, and bryostatin-1.

In a preferred multimodal therapy, both chemotherapeutic drugs andcytokines are co-administered with an antibody or immunoconjugate. Thecytokines, chemotherapeutic drugs and antibody or immunoconjugate can beadministered in any order, or together.

Immunoconjugates can be formulated according to known methods to preparepharmaceutically useful compositions, whereby the immunoconjugate iscombined in a mixture with a pharmaceutically suitable excipient.Sterile phosphate-buffered saline is one example of a pharmaceuticallysuitable excipient. Other suitable excipients are well-known to those inthe art. See, for example, Ansel et al., PHARMACEUTICAL DOSAGE FORMS ANDDRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro(ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (MackPublishing Company 1990), and revised editions thereof.

The immunoconjugate of the present invention can be formulated forintravenous administration via, for example, bolus injection orcontinuous infusion. Preferably, the antibody of the present inventionis infused over a period of less than about 4 hours, and morepreferably, over a period of less than about 3 hours. For example, thefirst 25-50 mg could be infused within 30 minutes, preferably even 15min, and the remainder infused over the next 2-3 hrs. Formulations forinjection can be presented in unit dosage form, e.g., in ampoules or inmulti-dose containers, with an added preservative. The compositions cantake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic conjugate. Control releasepreparations can be prepared through the use of polymers to complex oradsorb the immunoconjugate. For example, biocompatible polymers includematrices of poly(ethylene-co-vinyl acetate) and matrices of apolyanhydride copolymer of a stearic acid dimer and sebacic acid.Sherwood et al., Bio/Technology 10: 1446 (1992). The rate of release ofan immunoconjugate or antibody from such a matrix depends upon themolecular weight of the immunoconjugate or antibody, the amount ofimmunoconjugate or antibody within the matrix, and the size of dispersedparticles. Saltzman et al., Biophys. J. 55: 163 (1989); Sherwood et al.,supra. Other solid dosage forms are described in Ansel et al.,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea& Febiger 1990), and German) (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES,18th Edition (Mack Publishing Company 1990), and revised editionsthereof.

The immunoconjugate may also be administered to a mammal subcutaneouslyor even by other parenteral routes. Moreover, the administration may beby continuous infusion or by single or multiple boluses. Preferably, theantibody is infused over a period of less than about 4 hours, and morepreferably, over a period of less than about 3 hours.

More generally, the dosage of an administered immunoconjugate for humanswill vary depending upon such factors as the patient's age, weight,height, sex, general medical condition and previous medical history. Itmay be desirable to provide the recipient with a dosage ofimmunoconjugate, antibody fusion protein that is in the range of fromabout 1 mg/kg to 25 mg/kg as a single intravenous infusion, although alower or higher dosage also may be administered as circumstancesdictate. A dosage of 1-20 mg/kg for a 70 kg patient, for example, is70-1,400 mg, or 41-824 mg/m² for a 1.7-m patient. The dosage may berepeated as needed, for example, once per week for 4-10 weeks, once perweek for 8 weeks, or once per week for 4 weeks. It may also be givenless frequently, such as every other week for several months, or monthlyor quarterly for many months, as needed in a maintenance therapy.

Alternatively, an antibody may be administered as one dosage every 2 or3 weeks, repeated for a total of at least 3 dosages. Or, the antibodiesmay be administered twice per week for 4-6 weeks. If the dosage islowered to approximately 200-300 mg/m² (340 mg per dosage for a 1.7-mpatient, or 4.9 mg/kg for a 70 kg patient), it may be administered onceor even twice weekly for 4 to 10 weeks. Alternatively, the dosageschedule may be decreased, namely every 2 or 3 weeks for 2-3 months. Ithas been determined, however, that even higher doses, such as 20 mg/kgonce weekly or once every 2-3 weeks can be administered by slow i.v.infusion, for repeated dosing cycles. The dosing schedule can optionallybe repeated at other intervals and dosage may be given through variousparenteral routes, with appropriate adjustment of the dose and schedule.

In preferred embodiments, the subject antibodies are of use for therapyof cancer. Examples of cancers include, but are not limited to,carcinoma, lymphoma, glioblastoma, melanoma, sarcoma, and leukemia,myeloma, or lymphoid malignancies. More particular examples of suchcancers are noted below and include: squamous cell cancer (e.g.,epithelial squamous cell cancer), Ewing sarcoma, Wilms tumor,astrocytomas, lung cancer including small-cell lung cancer, non-smallcell lung cancer, adenocarcinoma of the lung and squamous carcinoma ofthe lung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma multiforme, cervical cancer, ovarian cancer, liver cancer,bladder cancer, hepatoma, hepatocellular carcinoma, neuroendocrinetumors, medullary thyroid cancer, differentiated thyroid carcinoma,breast cancer, ovarian cancer, colon cancer, rectal cancer, endometrialcancer or uterine carcinoma, salivary gland carcinoma, kidney or renalcancer, prostate cancer, vulvar cancer, anal carcinoma, penilecarcinoma, as well as head-and-neck cancer. The term “cancer” includesprimary malignant cells or tumors (e.g., those whose cells have notmigrated to sites in the subject's body other than the site of theoriginal malignancy or tumor) and secondary malignant cells or tumors(e.g., those arising from metastasis, the migration of malignant cellsor tumor cells to secondary sites that are different from the site ofthe original tumor).

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia,Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult SoftTissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, AnalCancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer,Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the RenalPelvis and Ureter, Central Nervous System (Primary) Lymphoma, CentralNervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma,Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, ChildhoodCerebellar Astrocytoma, Childhood Cerebral Astrocytoma, ChildhoodExtracranial Germ Cell Tumors, Childhood Hodgkin's Disease, ChildhoodHodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma,Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, ChildhoodNon-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Primary Liver Cancer, ChildhoodRhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood VisualPathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, EndocrinePancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma,Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and RelatedTumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, GastricCancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, GermCell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Headand Neck Cancer, Hepatocellular Cancer, Hodgkin's Lymphoma,Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers,Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell PancreaticCancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and OralCavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders,Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, MalignantThymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic OccultPrimary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,Metastatic Squamous Neck Cancer, Multiple Myeloma, MultipleMyeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, MyelogenousLeukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavityand Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell LungCancer, Occult Primary Metastatic Squamous Neck Cancer, OropharyngealCancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant FibrousHistiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone,Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian LowMalignant Potential Tumor, Pancreatic Cancer, Paraproteinemias,Polycythemia vera, Parathyroid Cancer, Penile Cancer, Pheochromocytoma,Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary LiverCancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvisand Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary GlandCancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small CellLung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used totreat malignant or premalignant conditions and to prevent progression toa neoplastic or malignant state, including but not limited to thosedisorders described above. Such uses are indicated in conditions knownor suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BasicPathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be treated include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be treated include, butare not limited to, benign dysproliferative disorders (e.g., benigntumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps oradenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen'sdisease, Farmer's Skin, solar cheilitis, and solar keratosis.

In preferred embodiments, the method of the invention is used to inhibitgrowth, progression, and/or metastasis of cancers, in particular thoselisted above.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia(including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

Other Therapeutic Agents

A wide variety of therapeutic reagents can be administered concurrentlyor sequentially, or advantageously conjugated to the antibodies of theinvention, for example, drugs, toxins, oligonucleotides,immunomodulators, hormones, hormone antagonists, enzymes, enzymeinhibitors, radionuclides, angiogenesis inhibitors, etc. The therapeuticagents recited here are those agents that also are useful foradministration separately with an antibody as described above.Therapeutic agents include, for example, chemotherapeutic drugs such asvinca alkaloids, anthracyclines, gemcitabine, epipodophyllotoxins,taxanes, antimetabolites, alkylating agents, antibiotics, SN-38, COX-2inhibitors, antimitotics, anti-angiogenic and pro-apoptotic agents,particularly doxorubicin, methotrexate, taxol, CPT-11, camptothecans,proteosome inhibitors, mTOR inhibitors, HDAC inhibitors, tyrosine kinaseinhibitors, and others. Other useful cancer chemotherapeutic drugs foradministering concurrently or sequentially, or for the preparation ofimmunoconjugates and antibody fusion proteins include nitrogen mustards,alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, COX-2inhibitors, antimetabolites, pyrimidine analogs, purine analogs,platinum coordination complexes, mTOR inhibitors, tyrosine kinaseinhibitors, proteosome inhibitors, HDAC inhibitors, camptothecins,hormones, and the like. Suitable chemotherapeutic agents are describedin REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co.1995), and in GOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OFTHERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), as well asrevised editions of these publications. Other suitable chemotherapeuticagents, such as experimental drugs, are known to those of skill in theart.

In a preferred embodiment, conjugates of camptothecins and relatedcompounds, such as SN-38, may be conjugated to hPAM4, hRS7 or otheranti-pancreatic cancer antibodies, for example as disclosed in U.S.patent application Ser. No. 12/026,811, filed Feb. 6, 2008; and Ser. No.11/388,032, filed Mar. 23, 2006, the Examples section of each of whichis incorporated herein by reference. In another preferred embodiment,gemcitabine is administered to the subject in conjunction with ⁹⁰Y-hPAM4and/or SN-38-hRS7.

A toxin can be of animal, plant or microbial origin. A toxin, such asPseudomonas exotoxin, may also be complexed to or form the therapeuticagent portion of an immunoconjugate of the antibodies. Other toxinssuitably employed in the preparation of such conjugates or other fusionproteins, include ricin, abrin, ribonuclease (RNase), DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, onconase,gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonasendotoxin. See, for example, Pastan et al., Cell 47:641 (1986),Goldenberg, Calif.—A Cancer Journal for Clinicians 44:43 (1994), Sharkeyand Goldenberg, Calif.—A Cancer Journal for Clinicians 56:226 (2006).Additional toxins suitable for use are known to those of skill in theart and are disclosed in U.S. Pat. No. 6,077,499, the Examples sectionof which is incorporated herein by reference.

An immunomodulator, such as a cytokine, may also be conjugated to, orform the therapeutic agent portion of the immunoconjugate, or may beadministered with, but unconjugated to, an antibody or antibodyfragment. As used herein, the term “immunomodulator” includes acytokine, a lymphokine, a monokine, a stem cell growth factor, alymphotoxin, a hematopoietic factor, a colony stimulating factor (CSF),an interferon (IFN), parathyroid hormone, thyroxine, insulin,proinsulin, relaxin, prorelaxin, follicle stimulating hormone (FSH),thyroid stimulating hormone (TSH), luteinizing hormone (LH), hepaticgrowth factor, prostaglandin, fibroblast growth factor, prolactin,placental lactogen, OB protein, a transforming growth factor (TGF),TGF-α, TGF-β, insulin-like growth factor (ILGF), erythropoietin,thrombopoietin, tumor necrosis factor (TNF), TNF-α, TNF-β, amullerian-inhibiting substance, mouse gonadotropin-associated peptide,inhibin, activin, vascular endothelial growth factor, integrin,interleukin (IL), granulocyte-colony stimulating factor (G-CSF),granulocyte macrophage-colony stimulating factor (GM-CSF), interferon-α,interferon-β, interferon-γ, S1 factor, IL-1, IL-1cc, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18 IL-21 and IL-25, LIF, kit-ligand, FLT-3,angiostatin, thrombospondin, endostatin and LT, and the like.

The immunoconjugate may comprise one or more radioactive isotopes usefulfor treating diseased tissue. Particularly useful therapeuticradionuclides include, but are not limited to ¹¹¹In, ¹⁷⁷Lu, ²¹²Bi,²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag,⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb,²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm,¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, and ²¹¹Pb. The therapeutic radionuclidepreferably has a decay energy in the range of 20 to 6,000 keV,preferably in the ranges 60 to 200 keV for an Auger emitter, 100-2,500keV for a beta emitter, and 4,000-6,000 keV for an alpha emitter.Maximum decay energies of useful beta-particle-emitting nuclides arepreferably 20-5,000 keV, more preferably 100-4,000 keV, and mostpreferably 500-2,500 keV. Also preferred are radionuclides thatsubstantially decay with Auger-emitting particles. For example, Co-58,Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, I-125, Ho-161,Os-189m and Ir-192. Decay energies of useful beta-particle-emittingnuclides are preferably <1,000 keV, more preferably <100 keV, and mostpreferably <70 keV. Also preferred are radionuclides that substantiallydecay with generation of alpha-particles. Such radionuclides include,but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215,Bi-211, Ac-225, Fr-221, At-217, Bi-213 and Fm-255. Decay energies ofuseful alpha-particle-emitting radionuclides are preferably 2,000-10,000keV, more preferably 3,000-8,000 keV, and most preferably 4,000-7,000keV.

For example, ⁶⁷Cu, considered one of the more promising radioisotopesfor radioimmunotherapy due to its 61.5-hour half-life and abundantsupply of beta particles and gamma rays, can be conjugated to anantibody using the chelating agent,p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid (TETA). Chase,supra. Alternatively, ⁹⁰Y, which emits an energetic beta particle, canbe coupled to an antibody, antibody fragment or fusion protein, usingdiethylenetriaminepentaacetic acid (DTPA), or more preferably usingDOTA. Methods of conjugating ⁹⁰Y to antibodies or targetable constructsare known in the art and any such known methods may be used. (See, e.g.,U.S. Pat. No. 7,259,249, the Examples section of which is incorporatedherein by reference. See also Lindén et al., Clin Cancer Res.11:5215-22, 2005; Sharkey et al., J Nucl Med. 46:620-33, 2005; Sharkeyet al., J Nucl Med. 44:2000-18, 2003.)

Additional potential therapeutic radioisotopes include ¹¹C, ¹³N, ¹⁵O,⁷⁵Br, ¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ¹¹³In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru,¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm,¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵⁸Co,⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Au, ⁷⁶Br, ¹⁶⁹Yb, and the like.

In another embodiment, a radiosensitizer can be used in combination witha naked or conjugated antibody or antibody fragment. For example, theradiosensitizer can be used in combination with a radiolabeled antibodyor antibody fragment. The addition of the radiosensitizer can result inenhanced efficacy when compared to treatment with the radiolabeledantibody or antibody fragment alone. Radiosensitizers are described inD. M. Goldenberg (ed.), CANCER THERAPY WITH RADIOLABELED ANTIBODIES, CRCPress (1995). Other typical radionsensitizers of interest for use withthis technology include gemcitabine, 5-fluorouracil, and cisplatin, andhave been used in combination with external irradiation in the therapyof diverse cancers, including pancreatic cancer. Therefore, we havestudied the combination of gemcitabine at what is believed to beradiosensitizing doses (once weekly 200 mg/m² over 4 weeks) ofgemcitabine combined with fractionated doses of ⁹⁰Y-hPAM4, and haveobserved objective evidence of pancreatic cancer reduction after asingle cycle of this combination that proved to be well-tolerated (nograde 3-4 toxicities by NCI-CTC v. 3 standard).

Antibodies or fragments thereof that have a boron addend-loaded carrierfor thermal neutron activation therapy will normally be affected insimilar ways. However, it will be advantageous to wait untilnon-targeted immunoconjugate clears before neutron irradiation isperformed. Clearance can be accelerated using an anti-idiotypic antibodythat binds to the anti-pancreatic cancer antibody. See U.S. Pat. No.4,624,846 for a description of this general principle. For example,boron addends such as carboranes, can be attached to antibodies.Carboranes can be prepared with carboxyl functions on pendant sidechains, as is well-known in the art. Attachment of carboranes to acarrier, such as aminodextran, can be achieved by activation of thecarboxyl groups of the carboranes and condensation with amines on thecarrier. The intermediate conjugate is then conjugated to the antibody.After administration of the antibody conjugate, a boron addend isactivated by thermal neutron irradiation and converted to radioactiveatoms which decay by alpha-emission to produce highly toxic, short-rangeeffects.

Pharmaceutically Suitable Excipients

The antibodies, fragments thereof or immunoconjugates to be delivered toa subject can comprise one or more pharmaceutically suitable excipients,one or more additional ingredients, or some combination of these. Theantibody can be formulated according to known methods to preparepharmaceutically useful compositions, whereby the immunoconjugate iscombined in a mixture with a pharmaceutically suitable excipient.Sterile phosphate-buffered saline is one example of a pharmaceuticallysuitable excipient. Other suitable excipients are well-known to those inthe art. See, for example, Ansel et al., PHARMACEUTICAL DOSAGE FORMS ANDDRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro(ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (MackPublishing Company 1990), and revised editions thereof.

The immunoconjugate can be formulated for intravenous administrationvia, for example, bolus injection or continuous infusion. Formulationsfor injection can be presented in unit dosage form, e.g., in ampules orin multi-dose containers, with an added preservative. The compositionscan take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Kits

Various embodiments may concern kits containing components suitable fortreating or diagnosing diseased tissue in a patient. Exemplary kits maycontain at least one antibody, antigen binding fragment or fusionprotein as described herein. If the composition containing componentsfor administration is not formulated for delivery via the alimentarycanal, such as by oral delivery, a device capable of delivering the kitcomponents through some other route may be included. One type of device,for applications such as parenteral delivery, is a syringe that is usedto inject the composition into the body of a subject. Inhalation devicesmay also be used. In certain embodiments, an anti-pancreatic cancerantibody or antigen binding fragment thereof may be provided in the formof a prefilled syringe or autoinjection pen containing a sterile, liquidformulation or lyophilized preparation of antibody (e.g., Kivitz et al.,Clin. Ther. 2006, 28:1619-29).

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions for use of the kit.

EXAMPLES

The examples below are illustrative of embodiments of the currentinvention and are not limiting to the scope of the claims.

Example 1 Therapy of a Patient with Inoperable and Metastatic PancreaticCarcinoma

The humanized PAM4 (hPAM4) antibody was made and radiolabeled asdescribed in U.S. Pat. No. 7,282,567 (incorporated herein by referencein its entirety). Patient 118-001, CWG, was a 63-year-old man withStage-IV pancreatic adenocarcinoma with multiple liver metastases,diagnosed in November of 2007. He agreed to undertake combinedradioimmunotherapy and gemcitabine chemotherapy as a first treatmentstrategy, and was then given a first therapy cycle of 6.5 mCi/m² of⁹⁰Y-hPAM4, combined with 200 mg/m² gemcitabine, whereby the gemcitabinewas given once weekly on weeks 1-4 and ⁹⁰Y-hPAM4 was given once-weeklyon weeks 2-4 (3 doses). Two months later, the same therapy cycle wasrepeated, because no major toxicities were noted after the first cycle.Already 4 weeks after the first therapy cycle, CT evidence of areduction in the diameters of the primary tumor and 2 of the 3 livermetastases surprisingly was noted, and this was consistent withsignificant decreases in the SUV values of FDG-PET scans, with 3 of the4 tumors returning to normal background SUV levels at this time (FIG. 1and FIG. 2). The patient's pre-therapy CA-19.9 level of 1,297 dropped toa low level of 77, further supportive of the therapy being effective.Table 1 shows the effects of combined radioimmunotherapy with ⁹⁰Y-hPAM4and gemcitabine chemotherapy in this patient. It was surprising andunexpected that such low doses of the radionuclide conjugated to theantibody combined with such low, nontoxic, doses of gemcitabine showedsuch antitumor activity even after only a single course of this therapy.

TABLE 1 Effects of Combined Radioimmunotherapy with ⁹⁰Y-hPAM4 andGemcitabine Chemotherapy in Metastatic Pancreatic Carcinoma BaselineLongest 4 wk post-Tx Baseline Tumor Diameter Longest PET 4 wk post-TxLocation (cm) Diameter (cm) (SUV) PET (SUV) Pancreatic tail 4.5 4.3 9.24.2 (primary) L hepatic met 1.9 1.9 4.1 background R post hepatic 1.71.6 3.7 background met R central hepatic 1.9 1.2 3.2 background met

Example 2 Therapy of Pancreatic Cancer Xenografts with Gemcitabine and⁹⁰Y-Labeled Peptide Pretargeted Using TF10

Summary

⁹⁰Y-hPAM4 IgG is currently being examined in Phase I/II trials incombination with gemcitabine in patients with Stage III/IV pancreaticcancer. We disclose a new approach for pretargeting radionuclides thatis able to deliver a similar amount of radioactivity to pancreaticcancer xenografts, but with less hematological toxicity, which would bemore amenable for combination with gemcitabine. The TF10 bispecificantibody was made by the DNL technique as described below. Nude micebearing ˜0.4 cm³ sc CaPan1 human pancreatic cancer were administeredTF10, followed 1 day later with a ⁹⁰Y-labeled hapten-peptide (IMP-288).Various doses and schedules of gemcitabine were added to this treatment,and tumor progression monitored up to 28 weeks. 0.7 mCi PT-RAIT aloneproduced only a transient 60% loss in blood counts, and animals given0.9 mCi of PT-RAIT alone and 0.7 mCi PT-RAIT+6 mg gemcitabine (humanequivalent ˜1000 mg/m²) had no histological evidence of renal toxicityafter 9 months. A single dose of 0.25 or 0.5 mCi PT-RAIT alone cancompletely ablate 20% and 80% of the tumors, respectively. Monthlyfractionated PT-RAIT (0.25 mCi/dose given at the start of eachgemcitabine cycle) added to a standard gemcitabine regimen (6 mg wkly×3;1 wk off; repeat 3 times) significantly increased the median time fortumors to reach 3.0 cm³ over PT-RAIT alone. Other treatment plansexamining non-cytotoxic radiosensitizing dose regimens of gemcitabineadded to PT-RAIT also showed significant improvements in treatmentresponse over PT-RAIT alone. The results show that PT-RAIT is apromising new approach for treating pancreatic cancer. These dataindicate combining PT-RAIT with gemcitabine will enhance therapeuticresponses.

Methods

TF10 bispecific antibody was prepared as described below. Forpretargeting, TF10 was given to nude mice bearing the human pancreaticadenocarcinoma cell line, CaPan1. After allowing sufficient time forTF10 to clear from the blood (16 h), the radiolabeled divalentHSG-peptide was administered. The small molecular weight HSG-peptide(˜1.4 kD) clears within minutes from the blood, entering theextravascular space where it can bind to anti-HSG arm of the pretargetedTF10 bsMAb. Within a few hours, >80% of the radiolabeled HSG-peptide isexcreted in the urine, leaving the tumor localized peptide and a traceamount in the normal tissues.

Results

FIG. 3 illustrates the therapeutic activity derived from a singletreatment of established (˜0.4 cm³) CaPan1 tumors with 0.15 mCi of⁹⁰Y-hPAM4 IgG, or 0.25 or 0.50 mCi of TF10-pretargeted ⁹⁰Y-IMP-288.Similar anti-tumor activity was observed for the 0.5-mCi pretargeteddose vs. 0.15-mCi dose of the directly radiolabeled IgG, buthematological toxicity was severe at this level of the direct conjugate(not shown), while the pretargeted dose was only moderately toxic (notshown). Indeed, the MTD for pretargeting using 90Y-IMP-288 is at least0.9 mCi in nude mice.

FIG. 4 shows that the combination of gemcitabine and PT-RAIT has asynergistic effect on anti-tumor therapy. Human equivalent doses of 1000mg/m² (6 mg) of gemcitabine (GEM) were given intraperitoneally to miceweekly for 3 weeks, then after resting for 1 week, this regimen wasrepeated 2 twice. PT-RAIT (0.25 mCi of TF10-pretargeted ⁹⁰Y-IMP-288) wasgiven 1 day after the first GEM dose in each of the 3 cycles oftreatment. GEM alone had no significant impact on tumor progression(survival based on time to progress to 3.0 cm³). PT-RAIT alone improvedsurvival compared to untreated animals, but the combined GEM withPT-RAIT regimen increased the median survival by nearly 10 weeks.Because hematological toxicity is not dose-limiting for PT-RAIT, but itis one of the limitations for gemcitabine therapy, these studies suggestthat PT-RAIT could be added to a standard GEM therapy with the potentialfor enhanced responses. The significant synergistic effect ofgemcitabine plus PT-RAIT was surprising and unexpected.

A further study examined the effect of the timing of administration onthe potentiation of anti-tumor effect of gemcitabine plus PT-RAIT. Asingle 6-mg dose of GEM was given one day before or 1 day after 0.25 mCiof TF10-pretargeted ⁹⁰Y-IMP-288 (not shown). This study confirmed whatis already well known with GEM, i.e., radiosensitization is best givenin advance of the radiation. Percent survival of treated mice showedlittle difference in survival time between PT-RAIT alone and PT-RAITwith gemcitabine given 22 hours after the radiolabeled peptide. However,administration of gemcitabine 19 hours prior to PT-RAIT resulted in asubstantial increase in survival (not shown).

Single dose PT-RAIT (0.25 mCi) combined with cetuximab (1 mg weekly ip;7 weeks) or with cetuximab+GEM (6 mg weekly×3) in animals bearing CaPan1showed the GEM+cetuximab combination with PT-RAIT providing a betterinitial response (FIG. 5), but the response associated with justcetuximab alone added to PT-RAIT was encouraging (FIG. 5), since it wasas good or better than PT-RAIT+GEM. Because the overall survival in thisstudy was excellent, with only 2 tumors in each group progressingto >2.0 cm3 after 24 weeks when the study was terminated, these resultsindicate a potential role for cetuximab when added to PT-RAIT.

Example 3 Effect of Fractionated Pretargeted Radioimmunotherapy(PT-RAIT) for Pancreatic Cancer Therapy

We evaluated fractionated therapy with ⁹⁰Y-DOTA-di-HSG peptide (IMP-288)and TF10. Studies using TF10 and radiolabeled IMP-288 were performed innude mice bearing s.c. CaPan1 human pancreatic cancer xenografts,0.32-0.54 cm³. For therapy, TF10-pretargeted ⁹⁰Y-IMP-288 was given [A]once (0.6mCi on wk 0) or [B] fractionated (0.3 mCi on wks 0 and 1), [C](0.2 mCi on wks 0, 1 and 2) or [D] (0.2 mCi on wks 0, 1 and 4).

Tumor regression (>90%) was observed in the majority of mice, 9/10,10/10, 9/10 and 8/10 in groups [A], [B], [C] and [D], respectively. Ingroup [A], maximum tumor regression in 50% of the mice was reached at3.7 wks, compared to 6.1, 8.1 and 7.1 wks in [B], [C] and [D],respectively. Some tumors showed regrowth. At week 14, the besttherapeutic response was observed in the fractionated group (2×0.3 mCi),with 6/10 mice having no tumors (NT) compared to 3/10 in the 3×0.2 mCigroups and 1/10 in the 1×0.6mCi group. No major body weight loss wasobserved. Fractionated PT-RAIT provides another alternative for treatingpancreatic cancer with minimum toxicity.

Example 4 ⁹⁰Y-hPAM4 Radioimmunotherapy (RAIT) Plus RadiosensitizingGemcitabine (GEM) Treatment in Advanced Pancreatic Cancer (PC)

⁹⁰Y-hPAM4, a humanized antibody highly specific for PC, showed transientactivity in patients with advanced disease, and GEM enhanced RAIT inpreclinical studies. This study evaluated repeated treatment cycles of⁹⁰Y-hPAM4 plus GEM in patients with untreated, unresectable PC. The⁹⁰Y-dose was escalated by cohort, with patients repeating 4-wk cycles(once weekly 200 mg/m² GEM, ⁹⁰Y-hPAM4 once-weekly wks 2-4) untilprogression or unacceptable toxicity. Response assessments used CT,FDG-PET, and CA19.9 serum levels.

Of 8 patients (3F/5M, 56-72 y.o.) at the 1^(st) 2 dose levels (6.5 and9.0 mCi/m² ⁹⁰Y-hPAM4×3), hematologic toxicity has been transient Grade1-2. Two patients responded to initial treatment with FDG SUV and CA19.9decreases, and lesion regression by CT. Both patients continue in goodperformance status now after 9 and 11 mo. and after a total of 3 and 4cycles, respectively, without additional toxicity. A 3^(rd) patient witha stable response by PET and CT and decreases in CA19.9 levels afterinitial treatment is now undergoing a 2nd cycle. Four other patients hadearly disease progression and the remaining patient is still beingevaluated. Dose escalation is continuing after fractionated RAIT with⁹⁰Y-hPAM4 plus low-dose gemcitabine demonstrated therapeutic activity atthe initial ⁹⁰Y-dose levels, with minimal hematologic toxicity, evenafter 4 therapy cycles.

Example 5 PAM4 Antibody Binds to the Earliest Stages of PancreaticCancer

Immunohistochemistry studies were performed with PAM4 antibody. Resultsobtained with stained tissue sections showed no reaction of PAM4 withnormal pancreatic ducts, ductules and acinar tissues (not shown). Incontrast, use of the MA5 antibody applied to the same tissue samplesshowed diffuse positive staining of normal pancreatic ducts and acinartissue (not shown). In tissue sections with well differentiated ormoderately differentiated pancreatic adenocarcinoma, PAM4 staining waspositive, with mostly cytoplasmic staining but intensification of at thecell surface. Normal pancreatic tissue in the same tissue sections wasunstained.

Table 2 shows the results of immunohistochemical analysis with PAM4 MAbin pancreatic adenocarcinoma samples of various stages ofdifferentiation. Overall, there was an 87% detection rate for allpancreatic cancer samples, with 100% detection of well differentiatedand almost 90% detection of moderately differentiated pancreaticcancers.

TABLE 2 PAM4 Binding to Different Stages of Pancreatic Cancer Cancer nFocal Diffuse Total Well Diff. 13 2 11 13 (100%) Moderately Diff. 24 615 21 (88%) Poorly Diff. 18 5 9 14 (78%) Total 55 13 35 48 (87%)

Table 3 shows that PAM4 immunohistochemical staining also detected avery high percentage of precursor lesions of pancreatic cancer,including PanIn-1A to PanIN-3, IPMN (intraductal papillary mucinousneoplasms) and MCN (mucinous cystic neoplasms). Overall, PAM4 stainingdetected 89% of all pancreatic precursor lesions. These resultsdemonstrate that PAM4 antibody-based immunodetection is capable ofdetecting almost 90% of pancreatic cancers and precursor lesions by invitro analysis. PAM4 expression was observed in the earliest phases ofPanIN development. Intense staining was observed in IPMN and MCN samples(not shown). The PAM4 epitope was present at high concentrations(intense diffuse stain) in the great majority of pancreaticadenocarcinomas. PAM4 showed diffuse, intense reactivity with theearliest stages of pancreatic carcinoma precursor lesions, includingPanIN-1, IPMN and MCN, yet was non-reactive with normal pancreatictissue. Taken together, these results show that the PAM4 antibody isbinds with very high specificity to the earliest stages of pancreaticcancer development.

TABLE 3 PAM4 Banding to Precursor Lesions of Pancreatic Cancer n FocalDiffuse Total PanIn-1A 27 9 15 24 (89%) PanIn-1B 20 4 16 20 (100%)PanIn-2 11 6 4 10 (91%) PanIn-3 5 2 0  2 (40%) Total PanIn 63 21 35 56(89%) IPMN 36 6 25 31 (86%) MCN 27 3 22 25 (92%)

Example 6 Preparation of Dock-and-Lock (DNL) Constructs

DDD and AD Fusion Proteins

The DNL technique can be used to make dimers, trimers, tetramers,hexamers, etc. comprising virtually any antibodies or fragments thereofor other effector moieties. For certain preferred embodiments, IgGantibodies or Fab antibody fragments may be produced as fusion proteinscontaining either a dimerization and docking domain (DDD) or anchoringdomain (AD) sequence. Although in preferred embodiments the DDD and ADmoieties are produced as fusion proteins, the skilled artisan willrealize that other methods of conjugation, such as chemicalcross-linking, may be utilized within the scope of the claimed methodsand compositions.

Bispecific antibodies may be formed by combining a Fab-DDD fusionprotein of a first antibody with a Fab-AD fusion protein of a secondantibody. Alternatively, constructs may be made that combine IgG-ADfusion proteins with Fab-DDD fusion proteins. The technique is notlimiting and any protein or peptide of use may be produced as an AD orDDD fusion protein for incorporation into a DNL construct. Wherechemical cross-linking is utilized, the AD and DDD conjugates are notlimited to proteins or peptides and may comprise any molecule that maybe cross-linked to an AD or DDD sequence using any cross-linkingtechnique known in the art. In certain exemplary embodiments, apolyethylene glycol (PEG) or other polymeric moiety may be incorporatedinto a DNL construct, as described in further detail below.

For pretargeting applications, an antibody or fragment containing abinding site for an antigen associated with a diseased tissue, such as atumor-associated antigen (TAA), may be combined with a second antibodyor fragment that binds a hapten on a targetable construct, to which atherapeutic and/or diagnostic agent is attached. The DNL-basedbispecific antibody is administered to a subject, circulating antibodyis allowed to clear from the blood and localize to target tissue, andthe conjugated targetable construct is added and binds to the localizedantibody for diagnosis or therapy.

Independent transgenic cell lines may be developed for each Fab or IgGfusion protein. Once produced, the modules can be purified if desired ormaintained in the cell culture supernatant fluid. Following production,any DDD-fusion protein module can be combined with any AD-fusion proteinmodule to generate a bispecific DNL construct. For different types ofconstructs, different AD or DDD sequences may be utilized. Exemplary DDDand AD sequences are provided below.

DDD1: (SEQ ID NO: 13) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2:(SEQ ID NO: 14) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1:(SEQ ID NO: 15) QIFYLAKQIVDNAIQQA AD2: (SEQ ID NO: 16)CGQIEYLAKQIVDNAIQQAGC

The skilled artisan will realize that DDD1 and DDD2 comprise the DDDsequence of the human RIIα form of protein kinase A. However, inalternative embodiments, the DDD and AD moieties may be based on the DDDsequence of the human RIα form of protein kinase A and a correspondingAKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.

DDD3 (SEQ ID NO: 17) SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAKDDD3C (SEQ ID NO: 18)MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK AD3(SEQ ID NO: 19) CGFEELAWKIAKMIWSDVFQQGC

Expression Vectors

The plasmid vector pdHL2 has been used to produce a number of antibodiesand antibody-based constructs. See Gillies et al., J Immunol Methods(1989), 125:191-202; Losman et al., Cancer (Phila) (1997), 80:2660-6.The di-cistronic mammalian expression vector directs the synthesis ofthe heavy and light chains of IgG. The vector sequences are mostlyidentical for many different IgG-pdHL2 constructs, with the onlydifferences existing in the variable domain (VH and VL) sequences. Usingmolecular biology tools known to those skilled in the art, these IgGexpression vectors can be converted into Fab-DDD or Fab-AD expressionvectors. To generate Fab-DDD expression vectors, the coding sequencesfor the hinge, CH2 and CH3 domains of the heavy chain are replaced witha sequence encoding the first 4 residues of the hinge, a 14 residueGly-Ser linker and the first 44 residues of human RIIα (referred to asDDD1). To generate Fab-AD expression vectors, the sequences for thehinge, CH2 and CH3 domains of IgG are replaced with a sequence encodingthe first 4 residues of the hinge, a 15 residue Gly-Ser linker and a 17residue synthetic AD called AKAP-IS (referred to as AD1), which wasgenerated using bioinformatics and peptide array technology and shown tobind RIIα dimers with a very high affinity (0.4 nM). See Alto, et al.Proc. Natl. Acad. Sci., U.S.A (2003), 100:4445-50.

Two shuttle vectors were designed to facilitate the conversion ofIgG-pdHL2 vectors to either Fab-DDD1 or Fab-AD1 expression vectors, asdescribed below.

Preparation of CH1

The CH1 domain was amplified by PCR using the pdHL2 plasmid vector as atemplate. The left PCR primer consisted of the upstream (5′) end of theCH1 domain and a SacII restriction endonuclease site, which is 5′ of theCH1 coding sequence. The right primer consisted of the sequence codingfor the first 4 residues of the hinge (PKSC) followed by four glycinesand a serine, with the final two codons (GS) comprising a Bam HIrestriction site. The 410 bp PCR amplimer was cloned into the PGEMT® PCRcloning vector (PROMEGA®, Inc.) and clones were screened for inserts inthe T7 (5′) orientation.

A duplex oligonucleotide was synthesized to code for the amino acidsequence of DDD1 preceded by 11 residues of the linker peptide, with thefirst two codons comprising a BamHI restriction site. A stop codon andan EagI restriction site are appended to the 3′ end. The encodedpolypeptide sequence is shown below.

(SEQ ID NO: 20) GSGGGGSGGGGSHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

Two oligonucleotides, designated RIIA1-44 top and RIIA1-44 bottom, whichoverlap by 30 base pairs on their 3′ ends, were synthesized and combinedto comprise the central 154 base pairs of the 174 bp DDD1 sequence. Theoligonucleotides were annealed and subjected to a primer extensionreaction with Taq polymerase. Following primer extension, the duplex wasamplified by PCR. The amplimer was cloned into PGEMT® and screened forinserts in the T7 (5′) orientation.

A duplex oligonucleotide was synthesized to code for the amino acidsequence of AD1 preceded by 11 residues of the linker peptide with thefirst two codons comprising a BamHI restriction site. A stop codon andan EagI restriction site are appended to the 3′ end. The encodedpolypeptide sequence is shown below.

(SEQ ID NO: 21) GSGGGGSGGGGSQIEYLAKQIVDNAIQQA

Two complimentary overlapping oligonucleotides encoding the abovepeptide sequence, designated AKAP-IS Top and AKAP-IS Bottom, weresynthesized and annealed. The duplex was amplified by PCR. The amplimerwas cloned into the PGEMT® vector and screened for inserts in the T7(5′) orientation.

Ligating DDD1 with CH1

A 190 bp fragment encoding the DDD1 sequence was excised from PGEMT®with BamHI and NotI restriction enzymes and then ligated into the samesites in CH1-PGEMT® to generate the shuttle vector CH1-DDD1-PGEMT®.

Ligating AD1 with CH1

A 110 bp fragment containing the AD1 sequence was excised from PGEMT®with BamHI and NotI and then ligated into the same sites in CH1-PGEMT®to generate the shuttle vector CH1-AD1-PGEMT®.

Cloning CH1-DDD1 or CH1-AD1 into pdHL2-Based Vectors

With this modular design either CH1-DDD1 or CH1-AD1 can be incorporatedinto any IgG construct in the pdHL2 vector. The entire heavy chainconstant domain is replaced with one of the above constructs by removingthe SacII/EagI restriction fragment (CH1-CH3) from pdHL2 and replacingit with the SacII/EagI fragment of CH1-DDD1 or CH1-AD1, which is excisedfrom the respective pGemT shuttle vector.

Construction of h679-Fd-AD1-pdHL2

h679-Fd-AD1-pdHL2 is an expression vector for production of h679 Fabwith AD1 coupled to the carboxyl terminal end of the CH1 domain of theFd via a flexible Gly/Ser peptide spacer composed of 14 amino acidresidues. A pdHL2-based vector containing the variable domains of h679was converted to h679-Fd-AD1-pdHL2 by replacement of the SacII/EagIfragment with the CH1-AD1 fragment, which was excised from theCH1-AD1-SV3 shuttle vector with SacII and EagI.

Construction of C-DDD1-Fd-hMN-14-pdHL2

C-DDD1-Fd-hMN-14-pdHL2 is an expression vector for production of astable dimer that comprises two copies of a fusion proteinC-DDD1-Fab-hMN-14, in which DDD1 is linked to hMN-14 Fab at the carboxylterminus of CH1 via a flexible peptide spacer. The plasmid vectorhMN-14(I)-pdHL2, which has been used to produce hMN-14 IgG, wasconverted to C-DDD1-Fd-hMN-14-pdHL2 by digestion with SacII and EagIrestriction endonucleases to remove the CH1-CH3 domains and insertion ofthe CH1-DDD1 fragment, which was excised from the CH1-DDD1-SV3 shuttlevector with SacII and EagI.

The same technique has been utilized to produce plasmids for Fabexpression of a wide variety of known antibodies, such as hLL1, hLL2,hPAM4, hR1, hRS7, hMN-14, hMN-15, hA19, hA20 and many others. Generally,the antibody variable region coding sequences were present in a pdHL2expression vector and the expression vector was converted for productionof an AD- or DDD-fusion protein as described above. The AD- andDDD-fusion proteins comprising a Fab fragment of any of such antibodiesmay be combined, in an approximate ratio of two DDD-fusion proteins perone AD-fusion protein, to generate a trimeric DNL construct comprisingtwo Fab fragments of a first antibody and one Fab fragment of a secondantibody.

C-DDD2-Fd-hMN-14-pdHL2

C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for production ofC-DDD2-Fab-hMN-14, which possesses a dimerization and docking domainsequence of DDD2 appended to the carboxyl terminus of the Fd of hMN-14via a 14 amino acid residue Gly/Ser peptide linker. The fusion proteinsecreted is composed of two identical copies of hMN-14 Fab held togetherby non-covalent interaction of the DDD2 domains.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides, which comprise the coding sequence forpart of the linker peptide and residues 1-13 of DDD2, were madesynthetically. The oligonucleotides were annealed and phosphorylatedwith T4 PNK, resulting in overhangs on the 5′ and 3′ ends that arecompatible for ligation with DNA digested with the restrictionendonucleases BamHI and PstI, respectively.

The duplex DNA was ligated with the shuttle vector CH1-DDD1-PGEMT®,which was prepared by digestion with BamHI and PstI, to generate theshuttle vector CH1-DDD2-PGEMT®. A 507 bp fragment was excised fromCH1-DDD2-PGEMT® with SacII and EagI and ligated with the IgG expressionvector hMN-14(I)-pdHL2, which was prepared by digestion with SacII andEagI. The final expression construct was designatedC-DDD2-Fd-hMN-14-pdHL2. Similar techniques have been utilized togenerated DDD2-fusion proteins of the Fab fragments of a number ofdifferent humanized antibodies.

h679-Fd-AD2-pdHL2

h679-Fab-AD2, was designed to pair as B to C-DDD2-Fab-hMN-14 as A.h679-Fd-AD2-pdHL2 is an expression vector for the production ofh679-Fab-AD2, which possesses an anchoring domain sequence of AD2appended to the carboxyl terminal end of the CH1 domain via a 14 aminoacid residue Gly/Ser peptide linker. AD2 has one cysteine residuepreceding and another one following the anchor domain sequence of AD1.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides (AD2 Top and AD2 Bottom), which comprisethe coding sequence for AD2 and part of the linker sequence, were madesynthetically. The oligonucleotides were annealed and phosphorylatedwith T4 PNK, resulting in overhangs on the 5′ and 3′ ends that arecompatible for ligation with DNA digested with the restrictionendonucleases BamHI and SpeI, respectively.

The duplex DNA was ligated into the shuttle vector CH1-AD1-PGEMT®, whichwas prepared by digestion with BamHI and SpeI, to generate the shuttlevector CH1-AD2-PGEMT®. A 429 base pair fragment containing CH1 and AD2coding sequences was excised from the shuttle vector with SacII and EagIrestriction enzymes and ligated into h679-pdHL2 vector that prepared bydigestion with those same enzymes. The final expression vector ish679-Fd-AD2-pdHL2.

Generation of TF2 DNL Construct

A trimeric DNL construct designated TF2 was obtained by reactingC-DDD2-Fab-hMN-14 with h679-Fab-AD2. A pilot batch of TF2 was generatedwith >90% yield as follows. Protein L-purified C-DDD2-Fab-hMN-14 (200mg) was mixed with h679-Fab-AD2 (60 mg) at a 1.4:1 molar ratio. Thetotal protein concentration was 1.5 mg/ml in PBS containing 1 mM EDTA.Subsequent steps involved TCEP reduction, HIC chromatography, DMSOoxidation, and IMP 291 affinity chromatography. Before the addition ofTCEP, SE-HPLC did not show any evidence of a₂b formation. Addition of 5mM TCEP rapidly resulted in the formation of a₂b complex consistent witha 157 kDa protein expected for the binary structure. TF2 was purified tonear homogeneity by IMP 291 affinity chromatography (not shown). IMP 291is a synthetic peptide containing the HSG hapten to which the 679 Fabbinds (Rossi et al., 2005, Clin Cancer Res 11:7122s-29s). SE-HPLCanalysis of the IMP 291 unbound fraction demonstrated the removal of a₄,a₂ and free kappa chains from the product (not shown).

The functionality of TF2 was determined by BIACORE® assay. TF2,C-DDD1-hMN-14+h679-AD1 (used as a control sample of noncovalent a₂bcomplex), or C-DDD2-hMN-14+h679-AD2 (used as a control sample ofunreduced a₂ and b components) were diluted to 1 μg/ml (total protein)and passed over a sensorchip immobilized with HSG. The response for TF2was approximately two-fold that of the two control samples, indicatingthat only the h679-Fab-AD component in the control samples would bind toand remain on the sensorchip. Subsequent injections of WI2 IgG, ananti-idiotype antibody for hMN-14, demonstrated that only TF2 had aDDD-Fab-hMN-14 component that was tightly associated with h679-Fab-AD asindicated by an additional signal response. The additional increase ofresponse units resulting from the binding of WI2 to TF2 immobilized onthe sensorchip corresponded to two fully functional binding sites, eachcontributed by one subunit of C-DDD2-Fab-hMN-14. This was confirmed bythe ability of TF2 to bind two Fab fragments of WI2 (not shown).

Production of TF10 Bispecific Antibody

A similar protocol was used to generate a trimeric TF10 DNL construct,comprising two copies of a C-DDD2-Fab-hPAM4 and one copy ofC-AD2-Fab-679. The cancer-targeting antibody component in TF10 wasderived from hPAM4, a humanized anti-pancreatic cancer mucin MAb thathas been studied in detail as a radiolabeled MAb (e.g., Gold et al.,Clin. Cancer Res. 13: 7380-7387, 2007). The hapten-binding component wasderived from h679, a humanized anti-histaminyl-succinyl-glycine (HSG)MAb. The TF10 bispecific ([hPAM4]₂×h679) antibody was produced using themethod disclosed for production of the (anti CEA)₂×anti HSG bsAb TF2, asdescribed above. The TF10 construct bears two humanized PAM4 Fabs andone humanized 679 Fab.

The two fusion proteins (hPAM4-DDD and h679-AD2) were expressedindependently in stably transfected myeloma cells. The tissue culturesupernatant fluids were combined, resulting in a two-fold molar excessof hPAM4-DDD. The reaction mixture was incubated at room temperature for24 hours under mild reducing conditions using 1 mM reduced glutathione.Following reduction, the DNL reaction was completed by mild oxidationusing 2 mM oxidized glutathione. TF10 was isolated by affinitychromatography using IMP 291-affigel resin, which binds with highspecificity to the h679 Fab.

A full tissue histology and blood cell binding panel has been examinedfor hPAM4 IgG and for an anti-CEA×anti-HSG bsMAb that is enteringclinical trials. hPAM4 binding was restricted to very weak binding tothe urinary bladder and stomach in 1/3 specimens (no binding was seen invivo), and no binding to normal tissues was attributed to theanti-CEA×anti-HSG bsMAb. Furthermore, in vitro studies against celllines bearing the H1 and H2 histamine receptors showed no antagonisticor agonistic activity with the IMP 288 di-HSG peptide, and animalstudies in 2 different species showed no pharmacologic activity of thepeptide related to the histamine component at doses 20,000 times higherthan that used for imaging. Thus, the HSG-histamine derivative does nothave pharmacologic activity.

The skilled artisan will realize that the DNL techniques disclosed abovemay be used to produce complexes comprising any combination ofantibodies or immunoconjugates—for example, a radiolabeled hPAM4 and anSN-38 conjugated hRS7 DNL complex.

Example 7 Sequence Variants for DNL

In certain preferred embodiments, the AD and DDD sequences incorporatedinto the DNL complex comprise the amino acid sequences of AD1 or AD2 andDDD1 or DDD2, as discussed above. However, in alternative embodimentssequence variants of AD and/or DDD moieties may be utilized inconstruction of the DNL complexes. For example, there are only fourvariants of human PKA DDD sequences, corresponding to the DDD moietiesof PKA RIα, RIIα, RIβ and RIIβ. The RIIα DDD sequence is the basis ofDDD1 and DDD2 disclosed above. The four human PKA DDD sequences areshown below. The DDD sequence represents residues 1-44 of RIIα, 1-44 ofRIIβ, 12-61 of RIα and 13-66 of RIβ. (Note that the sequence of DDD1 ismodified slightly from the human PKA RIIα DDD moiety.)

PKA RIα (SEQ ID NO: 22)SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEAK PKA RIβ(SEQ ID NO: 23) SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEENRQILAPKA RIIα (SEQ ID NO: 24) SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQPKA RIIβ (SEQ ID NO: 25) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER

The structure-function relationships of the AD and DDD domains have beenthe subject of investigation. (See, e.g., Burns-Harnuro et al., 2005,Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38;Alto et al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker etal., 2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J400:493-99; Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al.,2006, Mol Cell 24:397-408, the entire text of each of which isincorporated herein by reference.)

For example, Kinderman et al. (2006) examined the crystal structure ofthe AD-DDD binding interaction and concluded that the human DDD sequencecontained a number of conserved amino acid residues that were importantin either dimer formation or AKAP binding, underlined in SEQ ID NO:13below. (See FIG. 1 of Kinderman et al., 2006, incorporated herein byreference.) The skilled artisan will realize that in designing sequencevariants of the DDD sequence, one would desirably avoid changing any ofthe underlined residues, while conservative amino acid substitutionsmight be made for residues that are less critical for dimerization andAKAP binding.

(SEQ ID NO: 13) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

Alto et al. (2003) performed a bioinformatic analysis of the AD sequenceof various AKAP proteins to design an RII selective AD sequence calledAKAP-IS (SEQ ID NO:15), with a binding constant for DDD of 0.4 nM. TheAKAP-IS sequence was designed as a peptide antagonist of AKAP binding toPKA. Residues in the AKAP-IS sequence where substitutions tended todecrease binding to DDD are underlined in SEQ ID NO:15. The skilledartisan will realize that in designing sequence variants of the ADsequence, one would desirably avoid changing any of the underlinedresidues, while conservative amino acid substitutions might be made forresidues that are less critical for DDD binding.

AKAP-IS sequence (SEQ ID NO: 15) QIEYLAKQIVDNAIQQA

Gold (2006) utilized crystallography and peptide screening to develop aSuperAKAP-IS sequence (SEQ ID NO:26), exhibiting a five order ofmagnitude higher selectivity for the RII isoform of PKA compared withthe RI isoform. Underlined residues indicate the positions of amino acidsubstitutions, relative to the AKAP-IS sequence, which increased bindingto the DDD moiety of RIIα. In this sequence, the N-terminal Q residue isnumbered as residue number 4 and the C-terminal A residue is residuenumber 20. Residues where substitutions could be made to affect theaffinity for RIIα were residues 8, 11, 15, 16, 18, 19 and 20 (Gold etal., 2006). It is contemplated that in certain alternative embodiments,the SuperAKAP-IS sequence may be substituted for the AKAP-IS AD moietysequence to prepare DNL constructs. Other alternative sequences thatmight be substituted for the AKAP-IS AD sequence are shown in SEQ IDNO:27-29. Substitutions relative to the AKAP-IS sequence are underlined.It is anticipated that, as with the AD2 sequence shown in SEQ ID NO:16,the AD moiety may also include the additional N-terminal residuescysteine and glycine and C-terminal residues glycine and cysteine.

SuperAKAP-IS (SEQ ID NO: 26) QIEYVAKQIVDYAIHQAAlternative AKAP sequences (SEQ ID NO: 27) QIEYKAKQIVDHAIHQA(SEQ ID NO: 28) QIEYHAKQIVDHAIHQA (SEQ ID NO: 29) QIEYVAKQIVDHAIHQAFIG. 2 of Gold et al. disclosed additional DDD-binding sequences from avariety of AKAP proteins, shown below.

RII-Specific AKAPs AKAP-KL (SEQ ID NO: 30) PLEYQAGLLVQNAIQQAI AKAP79(SEQ ID NO: 31) LLIETASSLVKNAIQLSI AKAP-Lbc (SEQ ID NO: 32)LIEEAASRIVDAVIEQVK RI-Specific AKAPs AKAPce (SEQ ID NO: 33)ALYQFADRFSELVISEAL RIAD (SEQ ID NO: 34) LEQVANQLADQIIKEAT PV38(SEQ ID NO: 35) FEELAWKIAKMIWSDVF Dual-Specificity AKAPs AKAP7(SEQ ID NO: 36) ELVRLSKRLVENAVLKAV MAP2D (SEQ ID NO: 37)TAEEVSARIVQVVTAEAV DAKAP1 (SEQ ID NO: 38) QIKQAAFQLISQVILEAT DAKAP2(SEQ ID NO: 39) LAWKIAKMIVSDVMQQ

Stokka et al. (2006) also developed peptide competitors of AKAP bindingto PKA, shown in SEQ ID NO:40-42. The peptide antagonists weredesignated as Ht31 (SEQ ID NO:40), RIAD (SEQ ID NO:41) and PV-38 (SEQ IDNO:42). The Ht-31 peptide exhibited a greater affinity for the RIIisoform of PKA, while the RIAD and PV-38 showed higher affinity for RI.

Ht31 (SEQ ID NO: 40) DLIEEAASRIVDAVIEQVKAAGAY RIAD (SEQ ID NO: 41)LEQYANQLADQIIKEATE PV-38 (SEQ ID NO: 42) FEELAWKIAKMIWSDVFQQC

Hundsrucker et al. (2006) developed still other peptide competitors forAKAP binding to PKA, with a binding constant as low as 0.4 nM to the DDDof the RII form of PKA. The sequences of various AKAP antagonisticpeptides are provided in Table 1 of Hundsrucker et al., reproduced inTable 4 below. AKAPIS represents a synthetic RII subunit-bindingpeptide. All other peptides are derived from the RII-binding domains ofthe indicated AKAPs.

TABLE 4 AKAP Peptide sequences Peptide Sequence AKAPISQIFYLAKQIVDNAIQQA (SEQ ID NO: 15) AKAPIS-PQIEYLAKQIPDNAIQQA (SEQ ID NO: 43) Ht31KGADLIEEAASRIVDAVIEQVKAAG (SEQ ID NO: 44) Ht31-PKGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO: 45) AKAP7δ-wt-pepPEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 46) AKAP7δ-L304T-pepPEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 47) AKAP7δ-L308D-pepPEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 48) AKAP7δ-P-pepPEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO: 49) AKAP7δ-PP-pepPEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO: 50) AKAP7δ-L314E-pepPEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO: 51) AKAP1-pepEEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO: 52) AKAP2-pepLVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO: 53) AKAP5-pepQYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO: 54) AKAP9-pepLEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO: 55) AKAP10-pepNTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 56) AKAP11-pepVNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 57) AKAP12-pepNGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 58) AKAP14-pepTQDKNYEDELTQVALALVEDVINYA (SEQ ID NO: 59) Rab32-pepETSAKDNINIEEAARFLVEKILVNH (SEQ ID NO: 60)

Residues that were highly conserved among the AD domains of differentAKAP proteins are indicated below by underlining with reference to theAKAP IS sequence (SEQ ID NO:15). The residues are the same as observedby Alto et al. (2003), with the addition of the C-terminal alanineresidue. (See FIG. 4 of Hundsrucker et al. (2006), incorporated hereinby reference.) The sequences of peptide antagonists with particularlyhigh affinities for the RII DDD sequence were those of AKAP-IS,AKAP7δ-wt-pep, AKAP7δ-L304T-pep and AKAP7δ-L308D-pep.

AKAP-IS (SEQ ID NO: 15) QIEYLAKQIVDNAIQQA

Carr et al. (2001) examined the degree of sequence homology betweendifferent AKAP-binding DDD sequences from human and non-human proteinsand identified residues in the DDD sequences that appeared to be themost highly conserved among different DDD moieties. These are indicatedbelow by underlining with reference to the human PKA RIIα DDD sequenceof SEQ ID NO:13. Residues that were particularly conserved are furtherindicated by italics. The residues overlap with, but are not identicalto those suggested by Kinderman et al. (2006) to be important forbinding to AKAP proteins. The skilled artisan will realize that indesigning sequence variants of DDD, it would be most preferred to avoidchanging the most conserved residues (italicized), and it would bepreferred to also avoid changing the conserved residues (underlined),while conservative amino acid substitutions may be considered forresidues that are neither underlined nor italicized.

(SEQ ID NO: 13) SHIQ IP P GL TELLQGYT V EVLR Q QP P DLVEFA VE YF TR LREA R A

The skilled artisan will realize that these and other amino acidsubstitutions in the antibody moiety or linker portions of the DNLconstructs may be utilized to enhance the therapeutic and/orpharmacokinetic properties of the resulting DNL constructs.

Example 8 Cytotoxicity of RS7 Immunoconjugates to Cancer Cells

The RS7 antibody was conjugated to ranpirnase to produce animmunoconjugate with very high toxicity to a variety of epithelial celllines. Ranpirnase (Rap) is a single-chain ribonuclease of 104 aminoacids originally isolated from the oocytes of Rana pipiens. Rap exhibitscytostatic and cytotoxic effects on a variety of tumor cell lines invitro, as well as antitumor activity in vivo. Rap enters cells viareceptor-mediated endocytosis and once internalized into the cytosol,selectively degrades tRNA, resulting in inhibition of protein synthesisand induction of apoptosis. Rap can be administered repeatedly topatients without an untoward immune response, with reversible renaltoxicity reported to be dose-limiting (Mikulski et al., J Clin Oncol2002; 20:274-81; Int J Oncol 1993; 3:57-64).

In the studies below, an immunoconjugate comprising Rap attached to RS7showed broad and potent anti-proliferative activity against diversehuman epithelial cancer cell lines in vitro, as well as a human lungcancer xenograft in vivo. The IgG-based immunotoxin, designated2L-Rap(Q)-hRS7, comprised Rap(Q) (a mutant form of Rap with the putativeN-glycosylation site removed) conjugated to hRS7. 2L-Rap(Q)-hRS7suppressed tumor growth in a prophylactic model of nude mice bearingCalu-3 human non-small cell lung cancer xenografts, with an increase inthe median survival time (MST) from 55 to 96 days (P<0.01). The resultsdemonstrated superior efficacy of 2L-Rap(Q)-hRS7 as a therapeutic forvarious Trop-2-expressing cancers, such as cervical, breast, colon,pancreatic, ovarian, and prostate cancers.

Methods

Cell Proliferation Assay

Tumor cells were seeded in 96-well plates (1×10⁴ cells per well) andincubated with test articles at 0.01 to 100 nM for 72 h. The number ofliving cells was then determined using the soluble tetrazolium salt, MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium],following the manufacturer's protocol. The data from the dose-responsecurves were analyzed using GraphPad Prism software to obtain EC50 values(the concentration at which 50% inhibition occurs).

Colony-Formation Assay

Tumor cells were trypsinized and plated in 60-mm dishes (1×10³ cells).Cells were treated with each test article and allowed to form colonies.Fresh media containing the test article were added every 4 days, andafter 2 weeks of incubation, colonies were fixed in 4% formaldehyde andstained with Giemsa. Colonies>50 cells were enumerated under amicroscope.

In Vivo Toxicity

Naïve BALB/c mice (female, 7 weeks old, Taconic Farms, Germantown, N.Y.)were injected intravenously with various doses of (Q)-hRS7 ranging from25 to 400 μg per mouse and were monitored daily for visible signs oftoxicity and body weight change. The maximum tolerated dose (MTD) wasdefined as the highest dose at which no deaths occurred and the bodyweight loss was 20% or less of pretreatment animal weight (approximately20 g). Animals that experienced toxic effects were euthanized.

Therapeutic Efficacy in Tumor-Bearing Mice

Female NCr homozygous athymic nu/nu mice of approximately 20 g (5 weeksold when received from Taconic Farms) were inoculated s.c. with 1×10⁷Calu-3 human NSCLC cells and monitored for tumor growth by calipermeasurements of length×width of the tumor. Tumor volume was calculatedas (L×W2)/2. Once tumors reached approximately 0.15 cm³ in size, theanimals were divided into treatment groups of five per group. Therapyconsisted of either a single i.v. injection of 50 μg of (Q)-hRS7 or twoinjections of 25 μg administered seven days apart. A control groupreceived saline. Animals were monitored daily for signs of toxicity andwere humanely euthanized and deemed to have succumbed to diseaseprogression if tumors reached greater than 2.0 cm³ in size or becameulcerated. Additionally, if mice lost more than 20% of initial bodyweight or otherwise became moribund, they were euthanized. Survival datawere analyzed using Kaplan-Meier plots (log-rank analysis) with GraphPadPrism software. Differences were considered statistically significant atP<0.05.

Results

Binding Analysis

The reactivity of (Q)-hRS7 with Trop-2-expressing cell lines wasinitially assessed by ELISA and demonstrated for PC-3 and Calu-3 (datanot shown), both yielding an apparent dissociation constant (K_(D))about two-fold higher than that of hRS7 (0.28 nM vs. 0.14 nM). Nobinding was observed for the Trop-2-negative 22Rv1. Subsequent studieswere performed by flow cytometry in a total of 10 Trop-2-expressing celllines, and the results (not shown), indicate that there was virtually nodifference in the binding property of (Q)-hRS7 from that of hRS7.

RNase Activity

The NTT assay measures inhibition of protein synthesis due to mRNAdegradation by RNase. (Q)-hRS7 and rRap exhibited comparable RNaseactivity in this cell-free assay (not shown). Using yeast tRNA assubstrate, we estimated the kcat/Km (10⁹ M⁻¹ s⁻¹) of rRap and (Q)-hRS7to be 4.10 (±0.42) and 1.98, respectively. Thus the catalytic efficiencyof (Q)-hRS7 based on the concentration of Rap is about 50% of rRap

In Vitro Cytotoxicity

Based on the results of an MTS assay, (Q)-hRS7 is most potent againstME-180, T-47D, MDA-MB-468, and Calu-3 (not shown), with EC50 values of1.5, 2.0, 3.8, and 8.5 nM, respectively. For those cell lines showingless than <50% growth inhibition at 100 nM of (Q)-hRS7 with the MTSassay, we performed colony-formation assays to confirm that (Q)-hRS7 wascytotoxic at 10 or 100 nM to DU-145, PC-3, MCF7, SK-BR-3, BxPC-3,Capan-1, and SK-OV-3 (not shown). In both assays, hRS7, rRap, and thecombination of hRS7 and rRap showed little, if any, toxicity at 100 nMin all the cell lines evaluated. The Trop-2-negative AsPC-1 wasresistant to (Q)-hRS7 in both assays.

Internalization and Subcellular Location

The internalization of (Q)-hRS7 into ME-180 cells was clearly observed(not shown). The distribution pattern of intracellular (Q)-hRS7 inME-180, as detected directly by FITC conjugated GAH or indirectly byPE-conjugated GAM via mouse anti-Rap IgG, appeared to be nearlyidentical, suggesting that (Q)-hRS7 remains intact following entry intothese cells (not shown).

Therapeutic Efficacy in Tumor-Bearing Mice

As shown in FIG. 6A, either treatment (single dose, 50 μg or two dosesof 25 μg given 5 days apart) with (Q)-hRS7 significantly inhibited thegrowth of Calu-3 xenografts compared to untreated controls (P<0.019),with the median survival time increased from 55 days to 96 days (P<0.01;FIG. 6B).

Discussion

The clinical advancement of antibody-targeted RNases of animal origin isrelatively moderate, with the majority developed for treatinghematological malignancies and the targeting components conferred bysome forms of scFv (Schirrmann et al., Exp Opin Biol Ther 2009;9:79-95). To date, antibody-targeted animal RNases have not beenevaluated in patients with any cancer.

Two difficulties noted in the clinical development of plant or microbialimmunotoxins are undesirable toxicity and immunogenicity. Normal tissuetoxicity observed with these immunotoxins includes vascular leaksyndrome (VLS), hemolytic uremic syndrome (HUS), and hepatotoxicity(Kreitman, BioDrugs 2009; 23:1-13). The structural motif (x)D(y)identified to be responsible for the binding of ricin A-chain or IL-2 toendothelial cells is absent in the native sequence of Rap(Q), and hRS7is not crossreactive with human endothelial cells. We therefore considerthe likelihood of (Q)-hRS7 causing VLS to be remote. The large size of(Q)-hRS7 (˜180 kDa), which poses a potential concern for less rapidpenetration of tumors (Yokota et al., Cancer Res 1992; 52:3402-8),should prevent its clearance via kidneys and mitigate the risk for HUS.As for hepatotoxicity, we note that BL22, a recombinant anti-CD22immunotoxin composed of the disulfide-stabilized Fv of RFB4 fused toPE38, and similar immunotoxins such as LMB-2 (anti-Tac(Fv)-PE38), alsohad a very low MTD in mice due to nonspecific liver toxicity, yet BL22has been reported to be safe and efficacious in clinical trials ofpatients with hairy-cell leukemia (Kreitman et al., N Engl J Med 2001;345:241-7). Thus, the dose-limiting hepatotoxicity commonly observed inmice may be rarely manifested in humans (Kreitman, BioDrugs 2009;23:1-13).

Most genetically-engineered immunotoxins that have been evaluated incancer patients induced a strong humoral immune response, which shortensthe serum half-life and prevents further administration. It is expectedthat (Q)-hRS7 will be less immunogenic, because it comprises the fusionof a humanized antibody to a toxin (Rap) that appears to induce littleantibody response in patients (Mikulski et al., J Clin Oncol 2002;20:274-81).

Although the in vitro potency of (Q)-hRS7 was found to vary amongTrop-2-expressing cell lines when measured by the 3-day MTS assay, thecytotoxicity of (Q)-hRS7 was unequivocally demonstrated at 10 nM for allcell lines using the 14-day colony-formation assay. In addition to itspotent cytotoxicity against diverse cancer cell lines in vitro, (Q)-hRS7was shown to be effective in inhibiting the growth of Calu-3 human lungcancer xenografts in nude mice, thus validating the antitumor activityand stability of (Q)-hRS7 in vivo and confirming the suitability ofadding Trop-2 to the current list of antigens on solid cancers targetedby immunotoxins (Kreitman, AAPS J 2006; 8:E532-51; Pastan et al., NatRev Cancer 2006; Pastan et al., Ann Rev Med 2007; 58:221-37; Schirrmannet al., Exp Opin Biol Ther 2009; 9:79-95).

In conclusion, we have demonstrated that an amphibian RNaserecombinantly fused with a humanized anti-Trop-2 antibody showsselective and potent cytotoxicity against a variety of epithelialcancers, both in vitro and in vivo.

Example 9 High Potency of a Rap-Anti-Trop-2 IgG DNL Construct AgainstCarcinomas

Using the DNL techniques described in the Examples above, an E1-Rap DNLconstruct, comprising hRS7-IgG-Ad2 (anti-Trop-2) linked to four copiesof Rap-DDD2 was produced and showed potent in vitro growth inhibitoryproperties against a variety of carcinoma cell lines (not shown). Inbreast (MDA-MB-468), cervical (ME-180), and pancreatic (BxPC-3 andCapan-1) tumor lines, all of which express high levels of Trop-2, E1-Rapwas very potent, showing EC₅₀ in the subnanomolar range (5 to 890 pM),which was 1,000- to 100.00-fold higher than untargeted Rap or thecombination of Rap and hRS7. In cell lines expressing moderate levels ofTrop-2, such as the three prostate cancer lines (PC-3, DU 145, andLNCaP), E1-Rap was less potent, but still showed EC₅₀ in the nanomolarrange (1 to 890 nM). The cell binding data obtained for these solidcancer cell lines suggest that the sensitivity of a cell line to E1-Rapappears to correlate with its Trop-2 expression on the cell surface. Notoxicity was observed for E1-Rap in the prostate cancer line, 22Rv1,which fails to bind hRS7. These results show the efficacy of E1-Rap as anew therapeutic for Trop-2-positive solid tumors, including breast,colon, stomach, lung, ovarian, endometrial, cervical, pancreatic, andprostatic carcinomas.

Example 10 Combining Antibody-Targeted Radiation (Radioimmunotherapy)and Antibody-SN-38 Conjugates (ADC) Improves Pancreatic Cancer Therapy

We previously reported effective anti-tumor activity in nude micebearing human pancreatic tumors with ⁹⁰Y-humanized PAM4 IgG (hPAM4;⁹⁰Y-clivatuzumab tetraxetan) that was enhanced when combined withgemcitabine (GEM) (Gold et al., Int J. Cancer 109:618-26, 2004; ClinCancer Res 9:3929 S-37S, 2003). These studies led to clinical testing offractionated ⁹⁰Y-hPAM4 IgG combined with GEM that is showing encouragingobjective responses. While GEM is known for its radiosensitizingability, alone it is not a very effective therapeutic agent forpancreatic cancer and its dose is limited by hematologic toxicity, whichis also limiting for ⁹⁰Y-hPAM4 IgG.

We have observed promising anti-tumor activity with an ADC composed ofhRS7 IgG (humanized anti-epithelial glycoprotein-1; EGP-1) and SN-38,the active component of irinotecan. This ADC is very well tolerated inmice (e.g., ≧60 mg), yet just 4.0 mg (0.5 mg, twice-weekly×4) issignificantly therapeutic. EGP-1 (Trop2) is also expressed in mostpancreatic cancers.

The present study examined combinations of ⁹⁰Y-hPAM4 IgG with RS7-SN-38in nude mice bearing 0.35 cm³ subcutaneous xenografts of the humanpancreatic cancer cell line, Capan-1. Mice (n=10) were treated with asingle dose of ⁹⁰Y-hPAM4 IgG alone (130 μCi, i.e., the maximum tolerateddose (MTD) or 75 μCi), with RS7-SN-38 alone (as above), or combinationsof the 2 agents at the two ⁹⁰Y-hPAM4 dose levels, with the first ADCinjection given the same day as the ⁹⁰Y-hPAM4. All treatments weretolerated, with <15% loss in body weight. Objective responses occurredin most animals, but they were more robust in both of the combinationgroups as compared to each agent given alone. All animals in the0.13-mCi ⁹⁰Y-hPAM4 IgG+hRS7-SN-38 group achieved a tumor-free statewithin 4 weeks, while other animals continued to have evidence ofpersistent disease. These studies provide the first evidence thatcombined radioimmunotherapy and ADC enhances efficacy at safe doses.

In the ongoing PAM4 clinical trials, a four week clinical treatmentcycle is performed. In week 1, subjects are administered a dose of¹¹¹In-hPAM4, followed at least 2 days later by gemcitabine dose. Inweeks 2, 3 and 4, subjects are administered a ⁹⁰Y-hPAM4 dose, followedat least 2 days later by gemcitabine (200 mg/m²). Escalation started at3×6.5 mCi/m². The maximum tolerated dose in front-line pancreatic cancerpatients was 3×15 mCi/m² (hematologic toxicity is dose-limiting). Of 22CT-assessable patients, the disease control rate (CR+PR+SD) was 68%,with 5 (23%) partial responses and 10 (45%) having stabilization as bestresponse by RECIST criteria.

Preparation of Antibody-Drug Conjugate (ADC)

The SN-38 conjugated hRS7 antibody was prepared as shown in FIG. 7 andaccording to previously described protocols (Moon et al. J Med Chem2008, 51:6916-6926; Govindan et al., Clin Cancer Res 2009.15:6052-6061). A reactive bifunctional derivative of SN-38 (CL2A-SN-38,FIG. 7A) was prepared as described in U.S. Patent Application Publ. No.20200204589 (the Examples section of which is incorporated herein byreference). The formula of CL2A-SN-38 is(maleimido-[x]-Lys-PABOCO-20-O—SN-38, where PAB is p-aminobenzyl and ‘x’contains a short PEG). The synthetic scheme for CL2A-SN-38 production isfurther illustrated in FIG. 7B. Following reduction of disulfide bondsin the antibody with TCEP, the CL2A-SN-38 is reacted with reducedantibody to generate the SN-38 conjugated RS7 (FIG. 7C).

⁹⁰Y-hPAM4 is prepared as previously described (Gold et al., Clin CancerRes 2003, 9:3929 S-37S; Gold et al., Int J Cancer 2004, 109:618-26).

Combination RAIT+ADC

The hRS7 is present in most epithelial cancers (lung, breast, prostate,ovarian, colorectal, pancreatic) and hRS7-SN-38 conjugates are beingexamined in various human cancer-mouse xenograft models. Initialclinical trials with ⁹⁰Y-hPAM4 IgG plus radiosensitizing amounts of GEMare encouraging, with evidence of tumor shrinkage or stable disease.However, therapy of pancreatic cancer is very challenging. Therefore, acombination therapy was examined to determine whether it would response.Specifically, administration of hRS7-SN-38 at effective, yet non-toxicdoses was combined with RAIT with ⁹⁰Y-hPAM4 IgG.

The results shown in FIG. 8 demonstrate that the combination ofhRS7-SN-38 with ⁹⁰Y-hPAM4 was more effective than either treatmentalone, or the sum of the individual treatments. At a dosage of 75 μCi⁹⁰Y-hPAM4, only 1 of 10 mice was tumor-free after 20 weeks of therapy,the same as observed with hRS7-SN-38 alone. However, the combination ofhRS7-SN-38 with ⁹⁰Y-hPAM4 resulted in 4 of 10 mice that were tumor-freeafter 20 weeks, and the remaining subjects showed substantial decreasein tumor volume compared with either treatment alone. At 130 μCi⁹⁰Y-hPAM4 the difference was even more striking, with 9 of 10 animalstumor-free in the combined therapy group compared to 5 of 10 in the RAITalone group. These data demonstrate the synergistic effect of thecombination of hRS7-SN-38 with ⁹⁰Y-hPAM4. RAIT+ADC significantlyimproved time to progression and increased the frequency of tumor-freetreatment.

FIG. 9 shows that the combination of ADC with hRS7-SN-38 added to theMTD of RAIT with ⁹⁰Y-hPAM4 had minimal additional toxicity, indicated bythe % weight loss of the animal in response to treatment.

The effect of different sequential treatments on tumor survival isillustrated in FIG. 10. The results indicate that the optimal effect isobtained when RAIT is administered first, followed by ADC. In contrast,when ADC is administered first followed by RAIT, there is a decrease inthe incidence of tumor-free animals. Neither unconjugated hPAM4 nor hRS7antibodies had anti-tumor activity when given alone.

Experiments were performed in which both the radionuclide and the drug(SN-38) were conjugated to the same (PAM4) antibody. FIG. 11 shows thata 0.5 mg dose of hPAM4-SN-38, given twice weekly for 8 weeks, wasrelatively effective at inhibiting tumor growth. However, addition of⁹⁰Y-hPAM4 either on the same day or else prior to ADC improved theincidence of tumor-free animals at 12 weeks. These results show thatRAIT and ADC treatments targeting the same antigen can be giventogether. Even though hPAM4 IgG does not internalize, hPAM4-SN-38conjugate controlled tumor growth longer than hRS7-SN-38 in this modelsystem (which expresses high levels of PAM4 antigen).

Example 11 Efficacy of SN-38 Conjugates of Different Antibodies

CL2A-SN-38 was conjugated to humanized antibodies, hRS7 (anti-EGP-1),hPAM4 (anti-mucin), hMN-14 (anti-CEACAM5), hLL2 (anti-CD22), and hA20(anti-CD20). The conjugates, with a mean SN-38/MAb substitution (MSR) of˜6, were evaluated in the Capan-1 and BxPC-3 pancreatic human tumorxenografts and the Ramos human lymphoma xenograft grown s.c. in femaleathymic nude mice. When the starting tumor sizes in animals reached 0.2to 0.3 cm³, specific and non-targeting control conjugates wereadministered i.p. in a twice-weekly×4 weeks schedule using 25 mg/kg,12.5 mg/kg, or 5 mg/kg of protein dose.

All of the SN-38 antibody conjugates showed efficacy when examined in anappropriate target cancer cell line (data not shown). The hMN-14-SN-38conjugate showed efficacy against LS 174T human colon carcinomaxenograft in nude mice (not shown). The hA20-SN-38 and hLL2 conjugatesshowed efficacy in against Ramos human lymphoma xenografts in nude mice(not shown). The hRS7-SN-38 and hPAM4-SN-38 conjugates showed efficacyagainst Capan-1 human pancreatic cancer xenografts in nude mice (notshown).

A comparison was performed of the efficacy of SN-38 conjugates of hRS7,hPAM4, hMN14 and the control hA20 antibodies against Capan-1 humanpancreatic cancer xenografts in nude mice. As shown in FIG. 12, the hRS7and hPAM4 conjugates of SN-38 showed the greatest efficacy against ahuman pancreatic cancer cell line.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the products, compositions,methods and processes of this invention. Thus, it is intended that thepresent invention cover such modifications and variations, provided theycome within the scope of the appended claims and their equivalents.

What is claimed is:
 1. A method of treating pancreatic cancer comprisinga) administering to a subject with pancreatic cancer an anti-Trop-2antibody or antigen-binding fragment thereof conjugated to achemotherapeutic drug; and b) administering to said subject ananti-MUC-5ac antibody or antigen-binding fragment thereof conjugated toa radionuclide, wherein the anti-MUC-5ac antibody or fragment thereofcomprises the light complementarity determining region (CDR) sequencesCDR1 (SASSSVSSSYLY, SEQ ID NO:1); CDR2 (STSNLAS, SEQ ID NO:2); and CDR3(HQWNRYPYT, SEQ ID NO:3); and the heavy chain CDR sequences CDR1 (SYVLH,SEQ ID NO:4); CDR2 (YINPYNDGTQYNEKFKG, SEQ ID NO:5) and CDR3(GFGGSYGFAY, SEQ ID NO:6).
 2. The method of claim 1, wherein theanti-Trop-2 antibody or antigen-binding fragment thereof binds to thesame epitope as an anti-Trop-2 antibody comprising the light chain CDRsequences CDR1 (KASQDVSIAVA, SEQ ID NO:7); CDR2 (SASYRYT, SEQ ID NO:8);and CDR3 (QQHYITPLT, SEQ ID NO:9) and the heavy chain CDR sequences CDR1(NYGMN, SEQ ID NO:10); CDR2 (WINTYTGEPTYTDDFKG, SEQ ID NO:11) and CDR3(GGFGSSYWYFDV, SEQ ID NO:12).
 3. The method of claim 1, wherein thechemotherapeutic drug is SN-38 and the radionuclide is ⁹⁰Y.
 4. Themethod of claim 1, wherein the chemotherapeutic drug is selected fromthe group consisting of nitrogen mustards, ethylenimine derivatives,alkyl sulfonates, nitrosoureas, gemcitabine, triazenes, folic acidanalogs, anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs,purine analogs, antibiotics, enzyme inhibitors, epipodophyllotoxins,platinum coordination complexes, vinca alkaloids, substituted ureas,methyl hydrazine derivatives, adrenocortical suppressants, hormoneantagonists, endostatin, taxols, camptothecins, SN-38, doxorubicin,doxorubicin analogs, antimetabolites, alkylating agents, antimitotics,anti-angiogenic agents, tyrosine kinase inhibitors, mTOR inhibitors,heat shock protein (HSP90) inhibitors, proteosome inhibitors, HDACinhibitors, pro-apoptotic agents, methotrexate and CPT-11.
 5. The methodof claim 1, wherein the radionuclide is selected from the groupconsisting of ¹¹C, ¹³N, ¹⁵O, ³²P, ³³P, ⁴⁷Sc, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe,⁶²Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁷Ga, ⁷⁵Br, ⁷⁵Se, ⁷⁵Se, ⁷⁶Br, ⁷⁷As, ⁷⁷Br, ^(80m)Br,⁸⁹Sr, ⁹⁰Y, ⁹⁵Ru, ⁹⁷Ru, ⁹⁹Mo, ^(99m)Tc, ^(103m)Rh, ¹⁰³Ru, ¹⁰⁵Rh, ¹⁰⁵Ru,¹⁰⁷Hg, ¹⁰⁹Pd, ¹⁰⁹Pt, ¹¹¹Ag, ¹¹¹In, ^(113m)In, ¹¹⁹Sb, ^(121m)Te,^(122m)Te, ¹²⁵I, ^(125m)Te, ¹²⁶I, ¹³¹I, ¹³³I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm,¹⁵²Dy, ¹⁵³Sm, ¹⁶¹Ho, ¹⁶¹Tb, ¹⁶⁵Tm, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁶⁹Er,¹⁶⁹Yb, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ^(189m)Os, ¹⁸⁹Re, ¹⁹²Ir, ¹⁹⁴Ir, ¹⁹⁷Pt,¹⁹⁸Au, ¹⁹⁹Au, ¹⁹⁹Au, ²⁰¹Tl, ²⁰³Hg, ²¹¹At, ²¹¹Bi, ²¹¹Pb, ²¹²Bi, ²¹²Pb,²¹³Bi, ²¹⁵Po, ²¹⁷At, ²¹⁹Rn, ²²¹Fr, ²²³Ra, ²¹Ac, ²²⁵Ac and ²⁵⁵Fm.
 6. Themethod of claim 1, wherein the anti-Trop-2 antibody or fragment thereofcomprises the light chain CDR sequences CDR1 (KASQDVSIAVA, SEQ ID NO:7);CDR2 (SASYRYT, SEQ ID NO:8); and CDR3 (QQHYITPLT, SEQ ID NO:9) and theheavy chain CDR sequences CDR1 (NYGMN, SEQ ID NO:10); CDR2(WINTYTGEPTYTDDFKG, SEQ ID NO:11) and CDR3 (GGFGSSYWYFDV, SEQ ID NO:12).7. The method of claim 1, wherein the anti-Trop-2 antibody or fragmentthereof and the anti-pancreatic cancer mucin antibody or fragmentthereof are administered sequentially or concurrently.
 8. The method ofclaim 1, wherein the anti-Trop-2 antibody or fragment thereof and theanti-pancreatic cancer mucin antibody or fragment thereof are conjugatedtogether.
 9. The method of claim 1, wherein the anti-Trop-2 antibody orfragment thereof and the anti-MUC-5ac antibody or fragment thereof arechimeric, humanized or human antibodies or fragments thereof.
 10. Themethod of claim 1, further comprising administering to the subject atherapeutic agent selected from the group consisting of achemotherapeutic drug, an immunomodulator, a hormone, a hormoneantagonist, an enzyme, an siRNA, an RNAi, a photoactive therapeuticagent, an anti-angiogenic agent, a pro-apoptotic agent, an antibody andan antigen-binding antibody fragment.
 11. The method of claim 10,wherein the immunomodulator is selected from the group consisting of acytokine, a lymphokine, a monokine, a stem cell growth factor, alymphotoxin, a hematopoietic factor, a colony stimulating factor (CSF),an interferon (IFN), parathyroid hormone, thyroxin, insulin, proinsulin,relaxin, prorelaxin, follicle stimulating hormone (FSH), thyroidstimulating hormone (TSH), luteinizing hormone (LH), hepatic growthfactor, prostaglandin, fibroblast growth factor, prolactin, placentallactogen, OB protein, a transforming growth factor (TGF), TGF-α, TGF-β,insulin-like growth factor (ILGF), erythropoietin, thrombopoietin,TNF-α, TNF-β, mouse gonadotropin-associated peptide, inhibin, activin,vascular endothelial growth factor, integrin, interleukin (IL),granulocyte-colony stimulating factor (G-CSF), granulocytemacrophage-colony stimulating factor (GM-CSF), interferon-α,interferon-β, interferon-γ, S1 factor, IL-1, IL-1cc, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18 IL-21 and IL-25, LIF, kit-ligand, FLT-3,angiostatin, thrombospondin and endostatin.
 12. The method of claim 10,wherein the chemotherapeutic drug is selected from the group consistingof nitrogen mustards, ethylenimine derivatives, alkyl sulfonates,nitrosoureas, gemcitabine, triazenes, folic acid analogs,anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purineanalogs, antibiotics, enzyme inhibitors, epipodophyllotoxins, platinumcoordination complexes, vinca alkaloids, substituted ureas, methylhydrazine derivatives, adrenocortical suppressants, hormone antagonists,endostatin, taxols, camptothecins, SN-38, doxorubicin, doxorubicinanalogs, antimetabolites, alkylating agents, antimitotics,anti-angiogenic agents, tyrosine kinase inhibitors, mTOR inhibitors,heat shock protein (HSP90) inhibitors, proteosome inhibitors, HDACinhibitors, pro-apoptotic agents, methotrexate and CPT-11.
 13. Themethod of claim 10, wherein the antibody or antigen-binding antibodyfragment binds to an antigen selected from the group consisting ofCA19.9, DUPAN2, SPAN1, Nd2, B72.3, CC49, Le^(a), Le(y), CEACAM5,CEACAM6, CSAp, MUC-1, MUC-2, MUC-3, MUC-4, MUC-5ac, MUC-16, MUC-17,HLA-DR, CD40, CD74, CD138, HER2/neu, EGFR, EGP-1, EGP-2, VEGF, P1GF,insulin-like growth factor, tenascin, platelet-derived growth factor,IL-6, bcl-2, K-ras, p53 and cMET.